1194 Citations
Studies of the intermediate filament IF structure are a prerequisite of understanding their function In addition the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs Over the years considerable progress has been made on the atomic structure of the elementary building block of all IFs the coiled-coil dimer Here we discuss the approaches methods and practices that have contributed to this advance With abundant genetic information on hand bioinformatics approaches give important insights into the dimer structure including the head and tail regions poorly assessable experimentally At the same ... More
Studies of the intermediate filament (IF) structure are a prerequisite of understanding their function. In addition, the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs. Over the years, considerable progress has been made on the atomic structure of the elementary building block of all IFs, the coiled-coil dimer. Here, we discuss the approaches, methods and practices that have contributed to this advance. With abundant genetic information on hand, bioinformatics approaches give important insights into the dimer structure, including the head and tail regions poorly assessable experimentally. At the same time, the most important contribution has been provided by X-ray crystallography. Following the “divide-and-conquer” approach, many fragments from several IF proteins could be crystallized and resolved to atomic resolution. We will systematically cover the main procedures of these crystallographic studies, suggest ways to maximize their efficiency, and also discuss the possible pitfalls and limitations. In addition, electron paramagnetic resonance with site-directed spin labeling was another method providing a major impact toward the understanding of the IF structure. Upon placing the spin labels into specific positions within the full-length protein, one can evaluate the proximity of the labels and their mobility. This makes it possible to make conclusions about the dimer structure in the coiled-coil region and beyond, as well as to explore the dimer–dimer contacts. Less
The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand With the upstream improvements the bottleneck shifted towards the downstream process New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules overcoming the current chromatographic bottlenecks Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules This review focuses in the role of these two methodologies in current ... More
The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups. Less
In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity To guarantee stable biopharmaceutical processes and safe formulations both phenomenons have to be avoided as these may hinder regular processing steps This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions For this purpose additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected These additives were PEG PEG glycerol glycine NaCl and ArgHCl Concentrated lysozyme and glucose oxidase solutions at pH and served as ... More
In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregates. Less
PRMT is the less-characterized member of the protein arginine methyltransferase family in terms of structure activity and cellular functions PRMT is a modular protein containing a catalytic Ado-Met-binding domain and unique Src homology domain that binds proteins with proline-rich motifs PRMT is involved in a variety of cellular processes and has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners PRMT has been demonstrated to have weak methyltransferase activity on a histone H substrate but its optimal substrates have not yet been identified To obtain insights into the function and activity of PRMT we solve several ... More
PRMT2 is the less-characterized member of the protein arginine methyltransferase family in terms of structure, activity, and cellular functions. PRMT2 is a modular protein containing a catalytic Ado-Met-binding domain and unique Src homology 3 domain that binds proteins with proline-rich motifs. PRMT2 is involved in a variety of cellular processes and has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners. PRMT2 has been demonstrated to have weak methyltransferase activity on a histone H4 substrate, but its optimal substrates have not yet been identified. To obtain insights into the function and activity of PRMT2, we solve several crystal structures of PRMT2 from two homologs (zebrafish and mouse) in complex with either the methylation product S-adenosyl-L-homocysteine or other compounds including the first synthetic PRMT2 inhibitor (Cp1) studied so far. We reveal that the N-terminal-containing SH3 module is disordered in the full-length crystal structures, and highlights idiosyncratic features of the PRMT2 active site. We identify a new nonhistone protein substrate belonging to the serine-/arginine-rich protein family which interacts with PRMT2 and we characterize six methylation sites by mass spectrometry. To better understand structural basis for Cp1 binding, we also solve the structure of the complex PRMT4:Cp1. We compare the inhibitor–protein interactions occurring in the PRMT2 and PRMT4 complex crystal structures and show that this compound inhibits efficiently PRMT2. These results are a first step toward a better understanding of PRMT2 substrate recognition and may accelerate the development of structure-based drug design of PRMT2 inhibitors. Less
Cancer cell metabolism is a complex dynamic network of regulated pathways Interrogation of this network would benefit from rapid sensitive techniques that are adaptable to high-throughput formats facilitating novel compound screening This requires assays that have minimal sample preparation and are adaptable to lower-volume -well formats and automation Here we describe bioluminescent glucose lactate glutamine and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells glycolysis and glutaminolysis The sensitivity pmol sample broad linear range M and wide dynamic range -fold are advantageous for measuring both extracellular and intracellular metabolites Importantly ... More
Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1–5 pmol/sample), broad linear range (0.1–100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor changes in glucose and glutamine consumption and lactate and glutamate secretion over time. Homogeneous formats of the lactate and glutamate assays were robust (Z′ = 0.6–0.9) and could be multiplexed with a real-time viability assay to generate internally controlled data. Screening a small-compound library with these assays resulted in the identification of both inhibitors and activators of lactate and glutamate production. Less
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators Here we report the generation of a quantitative interaction network directly linking human proteins to the AAA ATPase p an essential hexameric protein with multiple cellular functions We show that the high-affinity interacting protein ASPL efficiently promotes p hexamer disassembly resulting in the formation of stable p ASPL heterotetramers High-resolution structural and biochemical studies indicate that an extended UBX domain eUBX in ASPL is critical for p hexamer disassembly and facilitates the assembly of p ASPL heterotetramers This spontaneous process is accompanied by ... More
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity. Less
Cannabinoid receptor CB is the principal target of -tetrahydrocannabinol THC a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use CB is activated by endocannabinoids and is a promising therapeutic target for pain management inflammation obesity and substance abuse disorders Here we present the crystal structure of human CB in complex with AM a stabilizing antagonist synthesized and characterized for this structural study The structure of the CB -AM complex reveals key features of the receptor and critical interactions for antagonist binding In combination with functional studies and molecular modeling ... More
Cannabinoid receptor 1 (CB1) is the principal target of ?9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 � crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals. Less
Tn and Tn -like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity fitness islands They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins TnsE recognizes mobile plasmids through an association with the processivity clamp and a ' recessed DNA end during conjugal replication However the mechanism for the specific recognition of ' recessed DNA ends remains unclear Structural analyses of the C-terminal domain of TnsE identified a ... More
Tn7 and Tn7-like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity/fitness islands. They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins. TnsE recognizes mobile plasmids through an association with the processivity clamp and a 3' recessed DNA end during conjugal replication. However, the mechanism for the specific recognition of 3' recessed DNA ends remains unclear. Structural analyses of the C-terminal domain of TnsE identified a novel protein fold
including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structurebased analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. Less
including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structurebased analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. Less
Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase CutC has emerged as an important route for the production of trimethylamine a microbial metabolite associated with both human disease and biological methane production Here we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline Within an unexpectedly polar active site CutC orients choline through hydrogen bonding with a putative general base and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety These structural data along with ... More
Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production. Less
The colloidal stability of a protein solution during downstream processing formulation and storage is a key issue for the biopharmaceutical production process Thus knowledge about colloidal solution characteristics such as the tendency to form aggregates or high viscosity at various processing conditions is of interest This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples For this purpose the diffusion coefficient the protein phase behavior and the dynamic viscosity in various systems containing the model proteins -lactalbumin lysozyme and glucose oxidase were studied ... More
The colloidal stability of a protein solution during downstream processing, formulation, and storage is a key issue for the biopharmaceutical production process. Thus, knowledge about colloidal solution characteristics, such as the tendency to form aggregates or high viscosity, at various processing conditions is of interest. This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples. For this purpose, the diffusion coefficient, the protein phase behavior, and the dynamic viscosity in various systems containing the model proteins α-lactalbumin, lysozyme, and glucose oxidase were studied. Each of these experiments revealed a wide range of variations in protein interactions depending on protein type, protein concentration, pH, and the NaCl concentration. All these variations showed to be mirrored by changes in the apparent diffusion coefficient in the respective samples. Whereas stable samples with relatively low viscosity showed an almost linear dependence, the deviation from the concentration-dependent linearity indicated both an increase in the sample viscosity and probability of protein aggregation. This deviation of the apparent diffusion coefficient from concentration-dependent linearity was independent of protein type and solution properties for this study. Thus, this single parameter shows the potential to act as a prognostic tool for colloidal stability of protein solutions. Less
Human metapneumovirus hMPV is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age The hMPV fusion F glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen To facilitate structure-based vaccine design we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of by X-ray crystallography The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus hRSV In contrast significant differences were apparent ... More
Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses. Less
In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step Unwanted protein precipitation or crystallization can lead to column pipe or filter blocking In formulation the formation of aggregates can even be lethal when injected into the patient The typical methodology to illustrate protein phase states is the generation of protein phase diagrams Commonly protein phase behavior is shown in dependence of protein and precipitant concentration Despite using high-throughput methods for the generation of phase diagrams the time necessary to reach equilibrium is the bottleneck ... More
In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step. Unwanted protein precipitation or crystallization can lead to column, pipe or filter blocking. In formulation, the formation of aggregates can even be lethal when injected into the patient. The typical methodology to illustrate protein phase states is the generation of protein phase diagrams. Commonly, protein phase behavior is shown in dependence of protein and precipitant concentration. Despite using high-throughput methods for the generation of phase diagrams, the time necessary to reach equilibrium is the bottleneck. Faster methods to predict protein phase behavior are desirable. In this study, hydrophobic interaction chromatography retention times were correlated to crystal size and form. High-throughput thermal stability measurements (melting and aggregation temperatures), using an Optim®2 system, were successfully correlated to glucose isomerase stability. By using hydrophobic interaction chromatography and thermal stability determinations, glucose isomerase conformational and colloidal stability were successfully predicted for different salts in a specific pH range. Less
Methods and devices for cell-free sorting and cloning of nucleic acid libraries are provided herein
Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation However alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease Glycogen phosphorylase GP a key enzyme in glycogen metabolism catalyzes the rate-limiting step of glycogen mobilization Moreover the allosteric regulation of the three GP isozymes muscle liver and brain by metabolites and phosphorylation in response to hormonal signaling fine-tunes glycogenolysis to fulfill energetic and metabolic requirements Whereas the structures of muscle and liver GPs have been known for decades the structure of brain GP ... More
Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 �) and in complex with its allosteric activator AMP (3.4 �). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen. Less
Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures and most VacA activities are dependent on its capacity to oligomerize The -kDa secreted VacA protein can undergo limited proteolysis to yield two domains designated p and p The p domain is required for membrane channel formation and intracellular toxic activities and the p domain has an important role in mediating VacA binding to cells Previous studies showed that the p domain has a predominantly -helical structure ... More
Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly �-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the �-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the �-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly. Less
The farnesoid X receptor FXR is a nuclear receptor responsible for homeostasis of bile acids lipids and glucose Compounds that alter endogenous FXR signaling can be used as therapeutic candidates or identified as potentially hazardous compounds depending on exposure doses and health states Therefore there is an increasing need for high-throughput screening assays of FXR activity to profile large numbers of environmental chemicals and drugs This chapter describes a workflow of FXR modulator identification and characterization To identify compounds that modulate FXR transactivation at the cellular level we first screen compounds from the Tox K compound library in an FXR-driven ... More
The farnesoid X receptor (FXR) is a nuclear receptor responsible for homeostasis of bile acids, lipids, and glucose. Compounds that alter endogenous FXR signaling can be used as therapeutic candidates or identified as potentially hazardous compounds depending on exposure doses and health states. Therefore, there is an increasing need for high-throughput screening assays of FXR activity to profile large numbers of environmental chemicals and drugs. This chapter describes a workflow of FXR modulator identification and characterization. To identify compounds that modulate FXR transactivation at the cellular level, we first screen compounds from the Tox21 10 K compound library in an FXR-driven beta-lactamase reporter gene assay multiplexed with a cell viability assay in the same well of the 1536-well plates. The selected compounds are then tested biochemically for their ability to modulate FXR-coactivator binding interactions using a time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator assay. The assay results from the workflow can be used to prioritize compounds for more extensive investigations. Less
Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM Here we present the crystal structure of the C perfringens S SleM protein at SleM comprises an N-terminal catalytic domain that adopts an irregular -barrel fold that is common to GH family lysozymes plus a C-terminal fibronectin type III domain The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution A truncated form of SleM that lacks the FnIII domain ... More
Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination. SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM. Here, we present the crystal structure of the�C. perfringens�S40 SleM protein at 1.8 �. SleM comprises an N-terminal catalytic domain that adopts an irregular ?/?-barrel fold that is common to GH25 family lysozymes, plus a C-terminal fibronectin type III domain. The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution. A truncated form of SleM that lacks the FnIII domain shows reduced activity against spore sacculi indicating that this domain may have a role in facilitating the position of substrate with respect to the enzyme's active site. Less
Nonlinear optical methods such as second harmonic generation SHG and two-photon excited UV fluorescence TPE-UVF imaging are promising approaches to address bottlenecks in the membrane protein structure determination pipeline The general principles of SHG and TPE-UVF are discussed here along with instrument design considerations Comparisons to conventional methods in high throughput crystallization condition screening and crystal quality assessment prior to X-ray diffraction are also discussed
Chromosome integrity depends on DNA structure-specific processing complexes that resolve DNA entanglement between sister chromatids If left unresolved these entanglements can generate either chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis These bridge structures are defined by the presence of the PICH protein which interacts with the BEND protein in mitosis To obtain structural insights into PICH BEND complex formation at the atomic level their respective NTPR and BD domains were cloned overexpressed and crystallized using M ammonium sulfate as a precipitant at pH The protein complex readily formed large hexagonal crystals belonging to space group P ... More
Chromosome integrity depends on DNA structure-specific processing complexes that resolve DNA entanglement between sister chromatids. If left unresolved, these entanglements can generate either chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis. These bridge structures are defined by the presence of the PICH protein, which interacts with the BEND3 protein in mitosis. To obtain structural insights into PICH�BEND3 complex formation at the atomic level, their respective NTPR and BD1 domains were cloned, overexpressed and crystallized using 1.56 M ammonium sulfate as a precipitant at pH 7.0. The protein complex readily formed large hexagonal crystals belonging to space group P6122, with unit-cell parameters a = b = 47.28, c = 431.58 � and with one heterodimer in the asymmetric unit. A complete multiwavelength anomalous dispersion (MAD) data set extending to 2.2 � resolution was collected from a selenomethionine-labelled crystal at the Swiss Light Source. Less
A new approach is described to screen for protein nanocrystals based on the reversibility of crystallization Methods to characterize nanocrystals are in strong need to facilitate sample preparation for serial femtosecond X-ray nanocrystallography SFX SFX enables protein structure determination by collecting X-ray diffraction from nano- and microcrystals using a free electron laser This technique is especially valuable for challenging proteins as for example membrane proteins and is in general a powerful method to overcome the radiation damage problem and to perform time-resolved structure analysis Nanocrystal growth cannot be monitored with common methods used in protein crystallography as the resolution of ... More
A new approach is described to screen for protein nanocrystals based on the reversibility of crystallization. Methods to characterize nanocrystals are in strong need to facilitate sample preparation for serial femtosecond X-ray nanocrystallography (SFX). SFX enables protein structure determination by collecting X-ray diffraction from nano- and microcrystals using a free electron laser. This technique is especially valuable for challenging proteins as for example membrane proteins and is in general a powerful method to overcome the radiation damage problem and to perform time-resolved structure analysis. Nanocrystal growth cannot be monitored with common methods used in protein crystallography, as the resolution of bright field microscopy is not sufficient. A high-performance method to screen for nanocrystals is second order nonlinear imaging of chiral crystals (SONICC). However, the high cost prevents its use in every laboratory, and some protein nanocrystals may be �invisible� to SONICC. In this work using a crystallization robot and a common imaging system precipitation comprised of nanocrystals and precipitation caused by aggregated protein can be distinguished. Less
This is a series of investigations into the molecular basis of the evolution of new protein functions The broad objective of this work was to determine exactly how a series of single amino acid mutations typical of an evolutionary trajectory can result in dramatic changes in catalytic activity specificity and protein solubility Various strategies were employed to achieve this aim including analysis of existing literature concerning the various models and theories relating to molecular evolution protein crystallography extensive enzyme kinetics and thermodynamic analysis theoretical analysis of catalytic mechanisms and computational simulation of protein dynamics Three model systems were investigated the ... More
This is a series of investigations into the molecular basis of the evolution of new protein functions. The broad objective of this work was to determine exactly how a series of single amino acid mutations, typical of an evolutionary trajectory, can result in dramatic changes in catalytic activity, specificity and protein solubility. Various strategies were employed to achieve this aim, including analysis of existing literature concerning the various models and theories relating to molecular evolution, protein crystallography, extensive enzyme kinetics and thermodynamic analysis, theoretical analysis of catalytic mechanisms and computational simulation of protein dynamics. Three model systems were investigated: the de novo designed Kemp Eliminase (KE07), the metallo-β-lactamases NDM1 and VIM2, and the N-acylhomoserine lactonase AiiA. Based on these studies, I was able to identify three clear phenomena that are important in molecular evolution: first, preorganization of the active sites residues is essential for efficient catalysis; second, remote mutations are capable of causing quite drastic rearrangements to the active site and substrate binding site by modulating the conformational landscape of a protein; third, intramolecular epistasis, the way that mutations interact with each other and the sequence background that they are introduced to, can constrain evolutionary trajectories and make the evolutionary potential of a protein contingent on its starting sequence. In Chapters 3-5 I focus on KE07, performing detailed kinetic analysis of hydrogenated and deuterated substrate, which revealed entropy-enthalpy compensation in the improvement in activity as well as an unusual change in the kinetic properties in the middle of the evolutionary trajectory. This is followed by comprehensive structural analysis, which reveals the enzyme has evolved to adopt a completely unexpected active site configuration via remote mutations. Finally, using computational simulations and solution fluorescence spectroscopy, I confirm that the in crystallo and kinetic observations are consistent with the behaviour of the protein in solution. Chapter 6 consists of a manuscript that describes the effects of conformational tinkering on the N-acyl-homoserine lactonase AiiA, specifically how remote mutations can have dramatic effects on activity by modulating the conformation of the active site. My contribution to this work included crystal structures and molecular dynamics simulations. Finally, Chapter 7 is a second manuscript that focuses on evolutionary contingency: by examining two related subfamilies of the metallo-β-lactamases, NDM1 and VIM2 we show that the evolvability of each is constrained by intramolecular epistasis and contingent on the starting sequence. To achieve the same final goal (greater whole cell activity), NDM1 evolved higher activity, while VIM2 evolved greater solubility. The crystals structures that I solved revealed the structural basis for the enhanced activity in NDM1 and that enhanced solubility in VIM2 is a result of an unprecedented (for an enzyme) structural rearrangement where the two halves of the α/β sandwich metallo- β-lactamase protein fold have separated and rearranged in an domain-swapped dimer. Less
Extraordinary antibodies capable of near pan-neutralization of HIV- have been identified One of the broadest is antibody E which recognizes the membrane-proximal external region MPER of the HIV- envelope and neutralizes of circulating HIV- strains If delivered passively E might serve to prevent or treat HIV- infection Antibody E however is markedly less soluble than other antibodies Here we describe the use of both structural biology and somatic variation to develop optimized versions of E with increased solubility From the structure of E we identified a prominent hydrophobic patch reversion of four hydrophobic residues in this patch to their hydrophilic ... More
Extraordinary antibodies capable of near pan-neutralization of HIV-1 have been identified. One of the broadest is antibody 10E8, which recognizes the membrane-proximal external region (MPER) of the HIV-1 envelope and neutralizes >95% of circulating HIV-1 strains. If delivered passively, 10E8 might serve to prevent or treat HIV-1 infection. Antibody 10E8, however, is markedly less soluble than other antibodies. Here, we describe the use of both structural biology and somatic variation to develop optimized versions of 10E8 with increased solubility. From the structure of 10E8, we identified a prominent hydrophobic patch; reversion of four hydrophobic residues in this patch to their hydrophilic germ line counterparts resulted in an ~10-fold decrease in turbidity. We also used somatic variants of 10E8, identified previously by next-generation sequencing, to optimize heavy and light chains; this process yielded several improved variants. Of these, variant 10E8v4 with 26 changes versus the parent 10E8 was the most soluble, with a paratope we showed crystallographically to be virtually identical to that of 10E8, a potency on a panel of 200 HIV-1 isolates also similar to that of 10E8, and a half-life in rhesus macaques of ~10 days. An anomaly in 10E8v4 size exclusion chromatography that appeared to be related to conformational isomerization was resolved by engineering an interchain disulfide. Thus, by combining a structure-based approach with natural variation in potency and solubility from the 10E8 lineage, we successfully created variants of 10E8 which retained the potency and extraordinary neutralization breadth of the parent 10E8 but with substantially increased solubility. Less
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic-resolution structural data Diffraction-based techniques such as single-crystal X-ray crystallography electron microscopy and neutron diffraction are well established and they have paved the road to the stunning successes of modern-day structural biology The major advances achieved in the last years in all aspects of structural research including sample preparation crystallization the construction of synchrotron and spallation sources phasing approaches and high-speed computing and visualization now provide specialists and nonspecialists alike with a steady flow of molecular images of unprecedented detail The present unit ... More
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic-resolution structural data. Diffraction-based techniques such as single-crystal X-ray crystallography, electron microscopy, and neutron diffraction are well established and they have paved the road to the stunning successes of modern-day structural biology. The major advances achieved in the last 20 years in all aspects of structural research, including sample preparation, crystallization, the construction of synchrotron and spallation sources, phasing approaches, and high-speed computing and visualization, now provide specialists and nonspecialists alike with a steady flow of molecular images of unprecedented detail. The present unit combines a general overview of diffraction methods with a detailed description of the process of a single-crystal X-ray structure determination experiment, from chemical synthesis or expression to phasing and refinement, analysis, and quality control. For novices it may serve as a stepping-stone to more in-depth treatises of the individual topics. Readers relying on structural information for interpreting functional data may find it a useful consumer guide. Less
Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome an organelle that consists of a membrane-enveloped magnetic nanoparticle Magnetite formation and its properties are controlled by a specific set of proteins MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified Here we studied the relationship between the MamC magnetite-interaction loop MIL structure and its magnetite interaction using an inert biomineralization protein-MamC chimera Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces Additionally the MamC-MIL induces the formation of ... More
Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome, an organelle that consists of a membrane-enveloped magnetic nanoparticle. Magnetite formation and its properties are controlled by a specific set of proteins. MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified. Here, we studied the relationship between the MamC magnetite-interaction loop (MIL) structure and its magnetite interaction using an inert biomineralization protein-MamC chimera. Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces. Additionally, the MamC-MIL induces the formation of larger magnetite crystals compared to protein-free and inert biomineralization protein control experiments. We suggest that the connection between the MamC-MIL structure and the protein’s charged surfaces is crucial for magnetite binding and thus for the size control of the magnetite nanoparticles. Less
The microtubule MT cytoskeleton plays important roles in many cellular processes In vivo MT nucleation is controlled by the -tubulin ring complex TuRC a -MDa complex composed of -tubulin small complex TuSC subunits The mechanisms underlying the assembly of TuRC are largely unknown In yeast the conserved protein Spc p both stimulates the assembly of the TuRC and anchors the TuRC to the spindle pole body Using a quantitative in vitro FRET assay we show that TuRC assembly is critically dependent on the oligomerization state of Spc p with higher-order oligomers dramatically enhancing the stability of assembled TuRCs Our in ... More
The microtubule (MT) cytoskeleton plays important roles in many cellular processes. In vivo, MT nucleation is controlled by the γ-tubulin ring complex (γTuRC), a 2.1-MDa complex composed of γ-tubulin small complex (γTuSC) subunits. The mechanisms underlying the assembly of γTuRC are largely unknown. In yeast, the conserved protein Spc110p both stimulates the assembly of the γTuRC and anchors the γTuRC to the spindle pole body. Using a quantitative in vitro FRET assay, we show that γTuRC assembly is critically dependent on the oligomerization state of Spc110p, with higher-order oligomers dramatically enhancing the stability of assembled γTuRCs. Our in vitro findings were confirmed with a novel in vivo γTuSC recruitment assay. We conclude that precise spatial control over MT nucleation is achieved by coupling localization and higher-order oligomerization of the receptor for γTuRC. Less
Haemophilus influenzae is an obligate human commensal pathogen that requires haem for survival and can acquire it from several host haemoproteins including haemopexin The haem transport system from haem-haemopexin consists of HxuC a haem receptor and the two-partner-secretion system HxuB HxuA HxuA which is exposed at the cell surface is strictly required for haem acquisition from haemopexin HxuA forms complexes with haem-haemopexin leading to haem release and its capture by HxuC The key question is how HxuA liberates haem from haemopexin Here we solve crystal structures of HxuA alone and HxuA in complex with the N-terminal domain of haemopexin A ... More
Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin. Less
Oral agents targeting Janus-associated kinases JAKs are promising new agents in clinical development To better understand the relationship between JAK inhibition and biological outcome compounds targeting JAKs were evaluated in peripheral human whole blood To date these analyses are low throughput and costly Here we developed a robust -well high-throughput flow-based assay approach to screen small molecules for JAK STAT signaling inhibition in human whole blood This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement Further the automation technologies and process optimizations developed here overcame sample integrity handling and multiparametric ... More
Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors. Less
The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine precision medicine Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules As a consequence individual and tailored productions require generic approaches for a fast and dedicated purification process development In this article different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and ... More
The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine (precision medicine). Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules. As a consequence, individual and tailored productions require generic approaches for a fast and dedicated purification process development. In this article, different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies. Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and mechanistic modeling tools. Such approaches constitute a good foundation for fast and effective process development for new products and processes, but their full potential becomes obvious in a correlated combination. Thus, different combinatorial approaches are presented, which might become future directions in the biopharmaceutical industry. Less
LUX ARRHYTHMO LUX is a Myb-domain transcription factor that plays an important role in regulating the circadian clock Lux mutations cause severe clock defects and arrhythmia in constant light and dark In order to examine the molecular mechanisms underlying the function of LUX the DNA-binding Myb domain was cloned expressed and purified The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays EMSAs demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity In order to investigate the specificity determinants of protein DNA interactions the protein was co-crystallized with a -mer ... More
LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock. Lux mutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein�DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using the MeshAndCollect workflow available at the ESRF. Less
A-kinase anchoring proteins AKAPs interact with the dimerization docking D D domains of regulatory subunits of the ubiquitous protein kinase A PKA AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling Here we elucidated the structure of an extended PKA-binding domain of AKAP bound to the D D domain of the regulatory RIIa subunits of PKA We identified three hydrophilic anchor points in AKAP outside the core PKA-binding domain which mediate contacts with the D D domain Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA We ... More
A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18� bound to the D/D domain of the regulatory RIIa subunits of PKA. We identified three hydrophilic anchor points in AKAP18� outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. Less
The SCP2-thiolase-like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism
Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP -thiolase-like protein SLP which is characterized by having the YDCF thiolase sequence fingerprint of the C -C loop SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals including human Deletion of the Trypanosoma brucei SLP gene TbSLP increases the doubling time of procyclic T brucei and causes a -fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion The crystal structure ... More
Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Less
Cytokines serve as a major mechanism of communication between immune cells and are the functional molecules at the end of immune pathways Abnormalities in cytokines are involved in a wide variety of diseases including chronic inflammation autoimmune diseases and cancer Cytokines are not only direct targets of therapeutics but also important biomarkers for assessing drug efficacy and safety Traditionally enzyme-linked immunosorbent assays ELISA were most popular for identifying and quantifying cytokines However ELISA is expensive labor intensive and low throughput Here we report the development of a miniaturized Luminex Austin TX assay platform to establish a panel of high-throughput multiplexed ... More
Cytokines serve as a major mechanism of communication between immune cells and are the functional molecules at the end of immune pathways. Abnormalities in cytokines are involved in a wide variety of diseases, including chronic inflammation, autoimmune diseases, and cancer. Cytokines are not only direct targets of therapeutics but also important biomarkers for assessing drug efficacy and safety. Traditionally, enzyme-linked immunosorbent assays (ELISA) were most popular for identifying and quantifying cytokines. However, ELISA is expensive, labor intensive, and low throughput. Here, we report the development of a miniaturized Luminex (Austin, TX) assay platform to establish a panel of high-throughput, multiplexed assays for measuring cytokines in human whole blood. The miniaturized 384-well Luminex assay uses <25% of the assay reagents compared with the 96-well assay. The development and validation of the 384-well Luminex cytokine assays enabled high-throughput screening of compounds in primary cells using cytokines as physiologically relevant readouts. Furthermore, this miniaturized multiplexed technology platform allows for high-throughput biomarker profiling of biofluids from animal studies and patient samples for translational research. Less
Serial femtosecond X-ray crystallography SFX using an X-ray free electron laser XFEL is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins including G-protein coupled receptors GPCRs which often only produce microcrystals An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in Here we report the deposition of the XFEL data and provide further details on crystallization XFEL data collection and analysis structure determination and the validation of the structural model The rhodopsin-arrestin crystal structure solved with ... More
Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. Less
Fish and human cytochrome P P A catalyze both steroid a-hydroxylation and a -lyase reactions Fish P A catalyzes only a-hydroxylation Both enzymes are microsomal-type P s integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment the signal anchor sequence The presence of this N-terminal region renders expression of full-length proteins impossible or challenging For some proteins variable truncation of the signal anchor sequence precludes expression or results in poor expression levels To crystallize P A and A in order to gain insight into their different activities we used an alternative N-terminal sequence to boost expression ... More
Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17a-hydroxylation and 17a,20-lyase reactions. Fish P450 17A2 catalyzes only 17a-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins impossible or challenging. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Less
Serogroup B Neisseria meningitidis MenB is the cause of an acute potentially severe infection known as invasive meningococcal disease IMD with two peaks in disease incidence occurring among adolescents and young adults to years of age Bexsero is the first genome-derived vaccine against MenB and it has recently been approved in countries worldwide Neisserial adhesin A NadA a meningococcal trimeric autotransporter adhesin TAA that acts in adhesion to and invasion of host epithelial cells is one of the three protein antigens included in Bexsero The main aim of this work was to obtain detailed insights into the structure of NadA ... More
Serogroup B Neisseria meningitidis (MenB) is the cause of an acute, potentially severe infection, known as invasive meningococcal disease (IMD) with two peaks in disease incidence occurring among adolescents and young adults 16 to 21 years of age. Bexsero is the first genome-derived vaccine against MenB, and it has recently been approved in >35 countries worldwide. Neisserial adhesin A (NadA), a meningococcal trimeric autotransporter adhesin (TAA) that acts in adhesion to, and invasion of, host epithelial cells, is one of the three protein antigens included in Bexsero. The main aim of this work was to obtain detailed insights into the structure of NadA variant 3 (NadAv3), the vaccine variant, and into the molecular mechanisms governing its transcriptional regulation by NadR (Neisseria adhesin A Regulator). The amount of NadA exposed on the meningococcal surface influences the antibody-mediated serum bactericidal response measured in vitro, which in turn correlates with protection in immunized subjects. A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal disease may be underestimated. The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. The functional, biochemical and high-resolution structural characterization of NadR is presented in the first part of the thesis (Part One). These studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. These findings shed light on the regulation of a key virulence factor and vaccine antigen of this important human pathogen. In the second part of the thesis (Part Two), strategies involving both protein engineering and crystal manipulation to increase the likelihood of solving the crystal structure of NadAv3 are described. The first approach was the rational design of new constructs of NadAv3, based on the recently solved crystal structure of a close sequence variant (NadAv5). Then, a comprehensive set of biochemical, biophysical and structural techniques were applied to investigate all the generated NadAv3 constructs, aiming to faithfully represent its natural trimeric status, essential for reliable structural, functional and epitope mapping studies. The well-characterized trimeric NadAv3 constructs represented a set of high quality reagents which were validated as probes for functional studies and as a platform for continued attempts for protein crystallization. Mutagenesis studies and screenings to identify a new crystal form of NadAv3 were performed to improve crystal quality, ultimately allowing the collection of several high quality X-ray diffraction data sets; structure determination is ongoing. The atomic resolution structure of NadAv3 will help to understand its biological role as both an adhesin and a vaccine antigen. For example, the high resolution structure will enable epitope mapping studies using human antibodies and thus permit a 7 deeper understanding of the molecular determinants of antibody binding and protective epitopes. In addition, it will help to understand the molecular basis of host-pathogen interactions mediated by specific human cell receptors. Less
Eukaryotes contain a diverse tapestry of specialized metabolites many of which are of significant pharmaceutical and industrial importance to humans Nevertheless exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems Recent advances in genomics metabolomics phylogenomics and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools ranging from multiomics-guided candidate gene identification to in vitro ... More
Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes. Less
Currently macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection However crystal harvesting and processing largely depend on manual operations Here a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments In this approach the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process in many cases eliminating the use of cryoprotectant agents while crystal-soaking experiments are ... More
Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities. Less
Examples are shown of protein crystallization in and data collection from solutions sandwiched between thin polymer films using vapour-diffusion and batch methods The crystallization platform is optimal for both visualization and in situ data collection with the need for traditional harvesting being eliminated In wells constructed from the thinnest plastic and with a minimum of aqueous liquid flash-cooling to K is possible without significant ice formation and without any degradation in crystal quality The approach is simple it utilizes low-cost consumables but yields high-quality data with minimal sample intervention and with the very low levels of background X-ray scatter that ... More
Examples are shown of protein crystallization in, and data collection from, solutions sandwiched between thin polymer films using vapour-diffusion and batch methods. The crystallization platform is optimal for both visualization and in situ data collection, with the need for traditional harvesting being eliminated. In wells constructed from the thinnest plastic and with a minimum of aqueous liquid, flash-cooling to 100 K is possible without significant ice formation and without any degradation in crystal quality. The approach is simple; it utilizes low-cost consumables but yields high-quality data with minimal sample intervention and, with the very low levels of background X-ray scatter that are observed, is optimal for microcrystals. Less
Metal ions and metallocofactors play important roles in a broad range of biochemical reactions Accordingly it has been estimated that as much as of the proteome uses transition metal ions to carry out a variety of essential functions The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties the diversity of which arises as transition metals can adopt different redox states and geometries dictated by the identity of the metal and the protein environment The coupling of a metal ion with an organic framework in metallocofactors such as heme and cobalamin further expands the chemical functionality of ... More
Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25�50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV�vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein. Less
Targeting A has recently been the main objective in Alzheimer s disease therapeutic approaches Passive immunization trials have encountered undesirable side effects but the therapy remains a promising option A -x has not been previously considered as a targetfor AD immunotherapy Tg - mouse model has been recently established and validated as an advantageous research tool in AD The current work deals with A - and explores its potential as atarget while elucidating therapeutic mechanism and crystallizing the NT X Fab in complex with A - aiming to further reveal the structural basis of antibody target affinity In the present ... More
Targeting Aβ has recently been the main objective in Alzheimer�s disease therapeutic approaches. Passive immunization trials have encountered undesirable side effects but the therapy remains a promising option. A�4-x has not been previously considered as a targetfor AD immunotherapy. Tg4-42 mouse model has been recently established and validated as an advantageous research tool in AD. The current work deals with A�4-42 and explores its potential as atarget, while elucidating therapeutic mechanism and crystallizing the NT4X Fab in complex with A�4-19 aiming to further reveal the structural basis of antibody: target affinity. In the present work, the novel monoclonal antibody NT4X specifically reacts with N-truncated A� at position 4 of A�. It binds Ntruncated A� under native and denaturing conditions and rescues invitro toxicity of A�4-42 and that of pyroglutamate A�pE3-42. The Fab fragment of the antibody was also able to prevent the in vitro toxicitycaused by A�4-42 in rat primary cortical neuron cultures. A�4-42 intracerebroventricular injection into wildtype miceinduced a behavioral deficit, shown as a reduction in alteration rate in a Y-Maze, which was prevented using the NT4X. The Fab fragmentof the antibody, at a higher dosage, was also able to prevent the in vivo behavioral deficit in a replicate experiment. The Tg4-42 homozygous mouse model, expressed A�4-42 andallows for is intraneuronal accumulation. At 6 months of age, the model already exhibits 50% neuronal loss in the CA1 region of thehippocampus and severe reference memory deficits in a Morris water maze. Preventative passive immunotherapy with the NT4X antibody and its Fab fragment was able to mitigate neuron loss significantly and rescue spatial memory deficits as compared to an isotype controlgroup. Crystallization of the NT4X Fab in complex with A�4-19 has beensuccessful. Diffraction data has been collected at 2.8 �. Efforts to v resolve the crystal structure of the complex are ongoing. Issues with antibody-target engagement in terms of affinity, species and conformation of A� bound may be dealt with before going to a clinical setting, with the help of information arising from the crystal structure of the NT4X Fab: A�4-19 complex. Less
Live virus vaccines are a critical component of worldwide vaccination strategy for reducing disease burden but often require complex biological production processes that are sensitive to many different factors both known and often unknown Prior application of high throughput process development HTPD approaches to these processes has been hampered by a complex design space low throughput analytics and challenges inherent in biosafety level containment and asepsis in laboratory automation In we initiated a project with HighRes Biosolutions to design and install an integrated high throughput screening platform to enable HTPD for biosafety level upstream process development studies The system incorporates ... More
Live virus vaccines are a critical component of worldwide vaccination strategy for reducing disease burden but often require complex biological production processes that are sensitive to many different factors, both known and often unknown. Prior application of high‐throughput process development (HTPD) approaches to these processes has been hampered by a complex design space, low‐throughput analytics, and challenges inherent in biosafety level 2 containment and asepsis in laboratory automation. In 2013, we initiated a project with HighRes Biosolutions to design and install an integrated high‐throughput screening platform to enable HTPD for biosafety level 2 upstream process development studies. The system incorporates the necessary tools for performing cell and virus culture studies in microplates, as well as advanced analytical capabilities necessary for assessment of cell phenotype, product quality, and antigen yield. To date, we have applied this system to screen optimal media formulations and viral production conditions in support of two viral vaccine programs, with phenotypic assays performed as an integrated part of the workflow. This case study illustrates the power of HTPD in addressing large‐scale biological screening challenges by narrowing a vast design space and identifying parameter interactions in live virus production processes. Less
Protein crystallization is a major bottleneck of structure determination by X-ray crystallography hampering the process by years in some cases Numerous matrix screening trials using significant amounts of protein are often applied while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts Here we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial The device is made with cost-effective materials and is completely automated for efficient and economical experimentation In the developed device trials can be realized with ... More
Protein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts. Here, we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial. The device is made with cost-effective materials and is completely automated for efficient and economical experimentation. In the developed device, 170 trials can be realized with unique concentrations of protein and precipitant established by gradient generation and isolated by elastomeric valving for crystallization incubation. Moreover, this device can be further downscaled to smaller nanowell volumes and larger scale integration. The device was calibrated using a fluorescent dye and compared to a numerical model where concentrations of each trial can be quantified to establish crystallization phase diagrams. Using this device, we successfully crystallized lysozyme and C-phycocyanin, as visualized by compatible crystal imaging techniques such as bright-field microscopy, UV fluorescence, and second-order nonlinear imaging of chiral crystals. Concentrations yielding observed crystal formation were quantified and used to determine regions of the crystallization phase space for both proteins. Low sample consumption and compatibility with a variety of proteins and imaging techniques make this device a powerful tool for systematic crystallization studies. Less
Staphylococcus pseudintermedius is a leading cause of disease in dogs and zoonosis causes human infections Methicillin-resistant S pseudintermedius strains are emerging resembling the global health threat of S aureus Therefore it is increasingly important to characterize potential targets for intervention against S pseudintermedius Here FhuD an S pseudintermedius surface lipoprotein implicated in iron uptake was characterized It was found that FhuD bound ferrichrome in an iron-dependent manner which increased the thermostability of FhuD by C The crystal structure of ferrichrome-free FhuD was determined via molecular replacement at resolution FhuD exhibits the class III solute-binding protein SBP fold with a ligand-binding ... More
Staphylococcus pseudintermedius is a leading cause of disease in dogs, and zoonosis causes human infections. Methicillin-resistant S. pseudintermedius strains are emerging, resembling the global health threat of S. aureus. Therefore, it is increasingly important to characterize potential targets for intervention against S. pseudintermedius. Here, FhuD, an S. pseudintermedius surface lipoprotein implicated in iron uptake, was characterized. It was found that FhuD bound ferrichrome in an iron-dependent manner, which increased the thermostability of FhuD by >15�C. The crystal structure of ferrichrome-free FhuD was determined via molecular replacement at 1.6 � resolution. FhuD exhibits the class III solute-binding protein (SBP) fold, with a ligand-binding cavity between the N- and C-terminal lobes, which is here occupied by a PEG molecule. The two lobes of FhuD were oriented in a closed conformation. These results provide the first detailed structural characterization of FhuD, a potential therapeutic target of S. pseudintermedius. Less
The characterization of macromolecular samples at synchrotrons has traditionally been restricted to direct exposure to X-rays but beamline automation and diversification of the user community has led to the establishment of complementary characterization facilities off-line The Sample Preparation and Characterization SPC facility at the EMBL PETRA synchrotron provides synchrotron users access to a range of biophysical techniques for preliminary or parallel sample characterization to optimize sample usage at the beamlines Here we describe a sample pipeline from bench to beamline to assist successful structural characterization using small angle X-ray scattering SAXS or macromolecular X-ray crystallography MX The SPC has developed ... More
The characterization of macromolecular samples at synchrotrons has traditionally been restricted to direct exposure to X-rays, but beamline automation and diversification of the user community has led to the establishment of complementary characterization facilities off-line. The Sample Preparation and Characterization (SPC) facility at the EMBL@PETRA3 synchrotron provides synchrotron users access to a range of biophysical techniques for preliminary or parallel sample characterization, to optimize sample usage at the beamlines. Here we describe a sample pipeline from bench to beamline, to assist successful structural characterization using small angle X-ray scattering (SAXS) or macromolecular X-ray crystallography (MX). The SPC has developed a range of quality control protocols to assess incoming samples and to suggest optimization protocols. A high-throughput crystallization platform has been adapted to reach a broader user community, to include chemists and biologists that are not experts in structural biology. The SPC in combination with the beamline and computational facilities at EMBL Hamburg provide a full package of integrated facilities for structural biology and can serve as model for implementation of such resources for other infrastructures. Less
The aggregation of proteins became one of the major challenges in the development of biopharmaceu ticals since the formation of aggregates can affect drug quality and immunogenicity However aggregation mechanisms are highly complex and the investigation requires cost time and material intensive experi mental effort In the present work the predictive power of protein characteristics for the phase behavior of three different proteins which are very similar in size and structure was studied In particular the surface hydrophobicity zeta potential and conformational flexibility of human lysozyme lysozyme from chicken egg white and -lactalbumin at pH and were assessed and examined ... More
The aggregation of proteins became one of the major challenges in the development of biopharmaceuticals since the formation of aggregates can affect drug quality and immunogenicity. However, aggregation mechanisms are highly complex and the investigation requires cost, time, and material intensive experimental effort. In the present work, the predictive power of protein characteristics for the phase behavior of three different proteins which are very similar in size and structure was studied. In particular, the surface hydrophobicity, zeta potential, and conformational flexibility of human lysozyme, lysozyme from chicken egg white, and α-lactalbumin at pH 3, 5, 7, and 9 were assessed and examined for correlation with experimental stability studies focusing on protein phase behavior induced by sodium chloride and ammonium sulfate. The molecular dynamics (MD) simulation based study of the conformational flexibility without precipitants was able to identify highly flexible protein regions which could be associated to the less regular secondary structure elements and random coiled and terminal regions in particular. Conformational flexibility of the entire protein structure and protein surface hydrophobicity could be correlated to differing aggregation propensities among the studied proteins and could be identified to be applicable for prediction of protein phase behavior in aqueous solution without precipitants. For prediction of protein phase behavior and aggregation propensity in aqueous solution with precipitants, protein flexibility was further studied in dependency of salt concentration and species by means of human lysozyme. Even though the results of the salt dependent MD simulations could not be shown to be sufficient for prediction of salt depending phase behavior, this study revealed a more pronounced destabilizing effect of ammonium sulfate in comparison to sodium chloride and thus, was found to be in good agreement with theoretical considerations along the Hofmeister series as well as experimentally evaluated phase behavior. Less
Trimeric autotransporter adhesins TAAs on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins However AtaA a TAA in the nonpathogenic Acinetobacter sp strain Tol shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain TM and the passenger domain contains an N-terminal head N-terminal stalk C-terminal head Chead and C-terminal stalk Cstalk The Chead-Cstalk-TM fragment which is conserved in many Acinetobacter TAAs has by itself the head-stalk-anchor architecture of a complete TAA Here we show ... More
Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three �-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner �-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. Less
-Carboxyvanillate decarboxylase LigW catalyzes the conversion of -carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin This enzyme was shown to require Mn for catalytic activity and the kinetic constants for the decarboxylation of -carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK- kcat s and kcat Km M s and Novosphingobium aromaticivorans kcat s and kcat Km M s were determined The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site The structure of LigW from N aromaticivorans bound with the substrate analogue -nitrovanillate Kd nM was ... More
5-Carboxyvanillate decarboxylase (LigW) catalyzes the conversion of 5-carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin. This enzyme was shown to require Mn2+ for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK-6 (kcat = 2.2 s�1 and kcat/Km = 4.0 � 104 M�1 s�1) and Novosphingobium aromaticivorans (kcat = 27 s�1 and kcat/Km = 1.1 � 105 M�1 s�1) were determined. The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site. The structure of LigW from N. aromaticivorans, bound with the substrate analogue, 5-nitrovanillate (Kd = 5.0 nM), was determined to a resolution of 1.07 �. The structure of this complex shows a remarkable enzyme-induced distortion of the nitro-substituent out of the plane of the phenyl ring by approximately 23�. A chemical reaction mechanism for the decarboxylation of 5-carboxyvanillate by LigW was proposed on the basis of the high resolution X-ray structures determined in the presence ligands bound in the active site, mutation of active site residues, and the magnitude of the product isotope effect determined in a mixture of H2O and D2O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step. Less
X-ray transparent Microfluidics for Protein Crystallization and Biomineralization A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University Waltham Massachusetts by Achini Opathalage Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics We ... More
X-ray transparent Microfluidics for Protein Crystallization and Biomineralization A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University, Waltham, Massachusetts by Achini Opathalage Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions.In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 �. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3. Less
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry Proteins can either be soluble crystalline or precipitated Additionally liquid-liquid phase separation gelation and skin formation can occur A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed For lysozyme from chicken egg white human lysozyme glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values pH and Sodium chloride ammonium sulfate olyethylene glycol and polyethylene glycol were used as precipitants Crystallizing conditions could be found for lysozyme from chicken egg ... More
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid-liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, olyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study
of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any
protein solution.
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of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any
protein solution.
Less
In the past decade advances in structure determination with electron microscopy of organic beam sensitive materials have been significant The newly developed techniques triggered by new microscope systems and new cameras made it possible to acquire D structural information from these samples to a resolution which was impossible to achieve before Knowledge is required to improve structure solution and every aspect of the process involved from treatment of radiation sensitive materials sample preparation TEM imaging and diffraction systems all the way to how data must be interpreted In this thesis I explained multiple new techniques and methods developed by us ... More
In the past decade, advances in structure determination with electron microscopy of organic, beam sensitive, materials have been significant. The newly developed techniques, triggered by new microscope systems and new cameras, made it possible to acquire 3D structural information from these samples to a resolution which was impossible to achieve before. Knowledge is required to improve structure solution and every aspect of the process involved, from treatment of radiation sensitive materials, sample preparation, TEM imaging and diffraction systems all the way to how data must be interpreted. In this thesis I explained multiple new techniques and methods developed by us, using both new microscopes as well as a new type of detector: Timepix. I describe how these tools can help to overcome (what were) the most important problems and bottlenecks in detection of very low dose electron diffraction. Less