Daniel, Edward Managing macromolecular crystallographic data with a laboratory information management system Journal Article In: Structural Biology, 2024. @article{noKey,
title = {Managing macromolecular crystallographic data with a laboratory information management system},
author = {Daniel, Edward},
url = {https://journals.iucr.org/d/issues/2024/08/00/von5001/index.html},
doi = {https://doi.org/10.1107/S2059798324005680},
year = {2024},
date = {2024-06-12},
journal = {Structural Biology},
abstract = {Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes. A prerequisite for successful structure determination is diffraction-quality crystals, which may require extensive optimization of both the protein and the conditions, and hence projects can stretch over an extended period, with multiple users being involved. The workflow from crystallization and crystal treatment to deposition and publication is well defined, and therefore an electronic laboratory information management system (LIMS) is well suited to management of the data. Completion of the project requires key information on all the steps being available and this information should also be made available according to the FAIR principles. As crystallized samples are typically shipped between facilities, a key feature to be captured in the LIMS is the exchange of metadata between the crystallization facility of the home laboratory and, for example, synchrotron facilities. On completion, structures are deposited in the Protein Data Bank (PDB) and the LIMS can include the PDB code in its database, completing the chain of custody from crystallization to structure deposition and publication. A LIMS designed for macromolecular crystallography, IceBear, is available as a standalone installation and as a hosted service, and the implementation of key features for the capture of metadata in IceBear is discussed as an example.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes. A prerequisite for successful structure determination is diffraction-quality crystals, which may require extensive optimization of both the protein and the conditions, and hence projects can stretch over an extended period, with multiple users being involved. The workflow from crystallization and crystal treatment to deposition and publication is well defined, and therefore an electronic laboratory information management system (LIMS) is well suited to management of the data. Completion of the project requires key information on all the steps being available and this information should also be made available according to the FAIR principles. As crystallized samples are typically shipped between facilities, a key feature to be captured in the LIMS is the exchange of metadata between the crystallization facility of the home laboratory and, for example, synchrotron facilities. On completion, structures are deposited in the Protein Data Bank (PDB) and the LIMS can include the PDB code in its database, completing the chain of custody from crystallization to structure deposition and publication. A LIMS designed for macromolecular crystallography, IceBear, is available as a standalone installation and as a hosted service, and the implementation of key features for the capture of metadata in IceBear is discussed as an example. |
Sandy, James Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source Journal Article In: Biochemistry, 2024. @article{noKey,
title = {Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source},
author = {Sandy, James},
url = {https://www.jove.com/t/65964/crystallization-situ-room-temperature-data-collection-using},
doi = {10.3791/65964},
year = {2024},
date = {2024-03-07},
journal = {Biochemistry},
abstract = {Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60° rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60° rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface. |
Shaheen, Aqsa, Tariq, Anam Identification of additional mechanistically important residues in the multidrug transporter styMdtM of Salmonella Typhi Journal Article In: Journal of Biomolecular Structure and Dynamics, 2023. @article{noKey,
title = {Identification of additional mechanistically important residues in the multidrug transporter styMdtM of Salmonella Typhi},
author = {Shaheen, Aqsa, Tariq, Anam},
url = {https://www.tandfonline.com/doi/abs/10.1080/07391102.2023.2263882},
doi = {https://doi.org/10.1080/07391102.2023.2263882},
year = {2023},
date = {2023-10-02},
journal = {Journal of Biomolecular Structure and Dynamics},
abstract = {Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi. styMdtM (locus name; STY4874) is a multidrug efflux transporter of the major facilitator superfamily expressed in S. Typhi. Functional assays identified several residues important for its transport activity. Here, we used an AlphaFold model to identify additional residues for analysis by mutagenesis. Mutation of peripheral residue Cys185 had no effect on the structure or function of the transporter. However, substitution of channel-lining residues Tyr29 and Tyr231 completely abolished transport function. Finally, mutation of Gln294, which faces peripheral helices of the transporter, resulted in the loss of transport of some substrates. Crystallization studies yielded diffraction data for the wild-type protein at 4.5 Å resolution and allowed the unit cell parameters to be established as a = b = 64.3 Å, c = 245.4 Å, α = β = γ = 90°, in space group P4. Our studies represent a further stepping stone towards a mechanistic understanding of the clinically important multidrug transporter styMdtM.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi. styMdtM (locus name; STY4874) is a multidrug efflux transporter of the major facilitator superfamily expressed in S. Typhi. Functional assays identified several residues important for its transport activity. Here, we used an AlphaFold model to identify additional residues for analysis by mutagenesis. Mutation of peripheral residue Cys185 had no effect on the structure or function of the transporter. However, substitution of channel-lining residues Tyr29 and Tyr231 completely abolished transport function. Finally, mutation of Gln294, which faces peripheral helices of the transporter, resulted in the loss of transport of some substrates. Crystallization studies yielded diffraction data for the wild-type protein at 4.5 Å resolution and allowed the unit cell parameters to be established as a = b = 64.3 Å, c = 245.4 Å, α = β = γ = 90°, in space group P4. Our studies represent a further stepping stone towards a mechanistic understanding of the clinically important multidrug transporter styMdtM. |
Idman, Lukas Structural characterization of plant derived hdr enzyme in the MEP pathway Journal Article In: Thesis/Dissertation, 2023. @article{noKey,
title = {Structural characterization of plant derived hdr enzyme in the MEP pathway},
author = {Idman, Lukas},
url = {https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahUKEwj6-qLgpOuBAxXkUjUKHaCUBugQFnoECBEQAQ&url=http%3A%2F%2Fuu.diva-portal.org%2Fsmash%2Fget%2Fdiva2%3A1793514%2FFULLTEXT01.pdf&usg=AOvVaw0cXfGBjNORqemopZgnNBuB&opi=89978449},
doi = {null},
year = {2023},
date = {2023-08-31},
journal = {Thesis/Dissertation},
abstract = {Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) are synthesized
as the final step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway by (E)-4-
hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR) and serve as the fundamental
precursors in the biosynthesis of isoprenoids. Previous studies have determined distinct
activities among HDR homologous originating from the same woody plants. This study
aims to, via crystallization, determine the structure for two Picea abies HDR isoforms
to shed light on the observed variation in enzymatic activity. Crystals for both of the
HDr isoforms have been achieved in this study. However, time constraints have prevented
any further analysis, leaving their structures unresolved. Nonetheless, future endeavors
dedicated to exploring the HDR building upon these results are likely to result in solved
structures.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) are synthesized
as the final step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway by (E)-4-
hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR) and serve as the fundamental
precursors in the biosynthesis of isoprenoids. Previous studies have determined distinct
activities among HDR homologous originating from the same woody plants. This study
aims to, via crystallization, determine the structure for two Picea abies HDR isoforms
to shed light on the observed variation in enzymatic activity. Crystals for both of the
HDr isoforms have been achieved in this study. However, time constraints have prevented
any further analysis, leaving their structures unresolved. Nonetheless, future endeavors
dedicated to exploring the HDR building upon these results are likely to result in solved
structures. |
Mikolajek, Halina, Sanchez-Weatherby, Juan Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi Journal Article In: IUCRj, 2023. @article{noKey,
title = {Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi},
author = {Mikolajek, Halina, Sanchez-Weatherby, Juan},
url = {https://journals.iucr.org/m/issues/2023/04/00/lz5063/index.html},
doi = {10.1107/S2052252523003810},
year = {2023},
date = {2023-05-18},
journal = {IUCRj},
abstract = {The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention. |
Adolph, Ramona S.: Biochemical and structural characterization of the regulation of human Sirtuin 1 by small molecules and proteins Journal Article In: Thesis/Dissertation, 2023. @article{noKey,
title = {Biochemical and structural characterization of the regulation of human Sirtuin 1 by small molecules and proteins},
author = {Adolph, Ramona S.:},
url = {https://epub.uni-bayreuth.de/id/eprint/5024/},
doi = {Thesis},
year = {2023},
date = {2023-04-27},
journal = {Thesis/Dissertation},
abstract = {Sirtuins are NAD+-dependent lysine deacylases, which regulate various cell signaling pathways and are associated with lifespan extension through caloric restriction. The human isoforms Sirt1-7 are linked to diverse age-related diseases such as Alzheimer’s, Parkinson’s, but also cancer and AIDS. Sirtuins are therefore emerging targets for therapeutic approaches and regulating sirtuin activity contextually is important. While current pharmacological modulation of sirtuin activity is almost exclusively restricted to inhibitors, which often lack isoform-specificity, potency, or bioavailability, the physiological modulation of sirtuin activity by other proteins is incompletely understood. This study covers important and new aspects for the physiological and pharmacological regulation of human Sirtuin 1. Concerning regulation by small molecules, the activation of hSirt1 by dehydroabietic acid was characterized providing potential for pharmacological modulation with a new nature-derived activator scaffold. In addition, the proposed anti-tumor potential of tranilast was linked to inhibition of hSirt1. Regarding the physiological modulation of hSirt1 activity by other proteins, conflictive results were available for hAROS. Within this thesis, hAROS was validated as hSirt1 inhibitor. Furthermore, a stable core was suggested for the intrinsically disordered protein hAROS, which will be used for future interaction analyses. Conversely, an hSirt1-activity modulating effect for human Hic1 has not been described yet. Focusing on the interaction between hSirt1 and the BTB/POZ domain of Hic1, this study showed that the interaction is limited to the catalytic domain of hSirt1 and does not require phosphorylation of hSirt1, nor the presence of NAD+ or sirtuin substrate. Hic1-BTB might even have a dual and concentration-dependent effect on hSirt1 activity, which will be subject to future studies. In addition, the first crystal structure of Hic1 was solved demonstrating a typical BTB fold and offering an analysis of putative interacting regions with hSirt1. Finally, the region of hSirt1 interacting with HIV1-Tat was also identified as the catalytic domain and an additional in vitro inhibition of the structurally similar catalytic domains of hSirt2 and hSirt3 was observed. Tat binding to hSirt1-3 was competitive to the sirtuin substrate, but not to NAD+. In line with this observation, several complex structures of hSirt3 with Tat peptides could demonstrate that acetylated or deacetylated Tat binds to the sirtuin substrate binding cleft with its basic region. Using structural superpositions and crosslinking, the major contribution to binding was found to be mediated by HIV1-Tat amino acids 49-52, while Tat amino acids 53-59 provide the additional high potency needed for physiological inhibition of hSirt1 through disruption of an important salt bridge between the hSirt1 SBD and catalytic core.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Sirtuins are NAD+-dependent lysine deacylases, which regulate various cell signaling pathways and are associated with lifespan extension through caloric restriction. The human isoforms Sirt1-7 are linked to diverse age-related diseases such as Alzheimer’s, Parkinson’s, but also cancer and AIDS. Sirtuins are therefore emerging targets for therapeutic approaches and regulating sirtuin activity contextually is important. While current pharmacological modulation of sirtuin activity is almost exclusively restricted to inhibitors, which often lack isoform-specificity, potency, or bioavailability, the physiological modulation of sirtuin activity by other proteins is incompletely understood. This study covers important and new aspects for the physiological and pharmacological regulation of human Sirtuin 1. Concerning regulation by small molecules, the activation of hSirt1 by dehydroabietic acid was characterized providing potential for pharmacological modulation with a new nature-derived activator scaffold. In addition, the proposed anti-tumor potential of tranilast was linked to inhibition of hSirt1. Regarding the physiological modulation of hSirt1 activity by other proteins, conflictive results were available for hAROS. Within this thesis, hAROS was validated as hSirt1 inhibitor. Furthermore, a stable core was suggested for the intrinsically disordered protein hAROS, which will be used for future interaction analyses. Conversely, an hSirt1-activity modulating effect for human Hic1 has not been described yet. Focusing on the interaction between hSirt1 and the BTB/POZ domain of Hic1, this study showed that the interaction is limited to the catalytic domain of hSirt1 and does not require phosphorylation of hSirt1, nor the presence of NAD+ or sirtuin substrate. Hic1-BTB might even have a dual and concentration-dependent effect on hSirt1 activity, which will be subject to future studies. In addition, the first crystal structure of Hic1 was solved demonstrating a typical BTB fold and offering an analysis of putative interacting regions with hSirt1. Finally, the region of hSirt1 interacting with HIV1-Tat was also identified as the catalytic domain and an additional in vitro inhibition of the structurally similar catalytic domains of hSirt2 and hSirt3 was observed. Tat binding to hSirt1-3 was competitive to the sirtuin substrate, but not to NAD+. In line with this observation, several complex structures of hSirt3 with Tat peptides could demonstrate that acetylated or deacetylated Tat binds to the sirtuin substrate binding cleft with its basic region. Using structural superpositions and crosslinking, the major contribution to binding was found to be mediated by HIV1-Tat amino acids 49-52, while Tat amino acids 53-59 provide the additional high potency needed for physiological inhibition of hSirt1 through disruption of an important salt bridge between the hSirt1 SBD and catalytic core. |
Larson, Grimm Structural and functional elucidation of four putative PQS-binding proteins in Pseudomonas aeruginosa Journal Article In: Thesis/ Dissertation, 2023. @article{noKey,
title = {Structural and functional elucidation of four putative PQS-binding proteins in Pseudomonas aeruginosa},
author = {Larson, Grimm},
url = {https://www.repository.cam.ac.uk/items/20d043a0-8135-41e6-a56a-aff2cd5aa229},
doi = {https://doi.org/10.17863/CAM.101975},
year = {2023},
date = {2023-03-31},
journal = {Thesis/ Dissertation},
abstract = {Pseudomonas aeruginosa is a multi-drug resistant, human opportunistic pathogen. If left untreated, P. aeruginosa can cause severe to life-threatening infections in people with burns, cystic fibrosis, and in immunocompromised patients. During chronic infections, P. aeruginosa primarily co-ordinates virulence in the host through a cell-to-cell communication mechanism called quorum sensing (QS). There are three key QS systems in P. aeruginosa responsible for driving global changes in virulence gene expression: the las, rhl, and pqs systems. Each of the las, rhl, and pqs systems rely on a receptor-autoinducer relationship: these receptor-autoinducer complexes are LasR-OdDHL, RhlR-BHL, and PqsR-PQS, respectively. When the receptors (LasR, RhlR, PqsR) bind with their cognate autoinducer (OdDHL, BHL, PQS, respectively), they act as transcription factors that ultimately stimulate the expression of hundreds of virulence-associated genes. The influence these QS systems have on the expression of virulence determinants has led to decades of scientific research focusing on the characterisation of these regulators. Although LasR and PqsR have been structurally elucidated, the RhlR crystal structure has long eluded characterisation and has been highly sought after due to its obvious potential as a therapeutic target.
In a collaborative research effort, I helped to identify ten additional proteins as putative binding partners of the pqs autoinducer, PQS. Four of the ten proteins identified were the cyanide synthase (HcnC), a putative protease (PfpI), a phenazine biosynthetic protein (PhzD1), and the QS regulator RhlR. For this PhD project, I aimed to structurally and biochemically characterise these four proteins to, in part, confirm their proposed interaction with PQS. A novel ligand (benzoic acid) was discovered bound in the active site of PhzD1 (crystal structure solved to 1.1 Å). Additionally, the crystal structure for PfpI was resolved at 1.4 Å resolution. The PfpI tertiary and quaternary structures obtained in this study suggested a possible role in electrophile detoxification, a hypothesis which I confirmed in vitro using 1D NMR. To complement the novel PfpI structural and biochemical data, I generated and confirmed “clean” pfpI deletion mutants for phenotypic and ‘omic analyses. I observed discrepancies in phenotypes between the pfpI deletion mutant and the pfpI transposon mutants previously reported in the published literature, which I sought to reconcile through subsequent whole genome sequencing (WGS) of these previously published strains. WGS of the pfpI transposon mutants revealed a plethora of unexpected mutations elsewhere in the genome, which likely contribute to many of the reported phenotypes. The “clean” deletion mutant that I generated harboured no significant additional mutations. Proteomic profiling of the pfpI deletion mutant exhibited altered protein expression in systems involved in Type VI secretion, motility, and metabolism.
Overall, the work presented in this dissertation further illustrates the intractability of purifying the QS transcriptional regulator, RhlR. I report benzoic acid to be a novel binding partner for the phenazine biosynthetic protein, PhzD1. Phenotypic analyses of pfpI mutants and consequent WGS highlight the need for rigorous strain validation when using transposon mutant libraries. Using the PfpI structural data I obtained during this study, I hypothesised and confirmed a novel detoxification role for PfpI in P. aeruginosa. Lastly, proteomic analysis of a pfpI deficient mutant revealed global dysregulation of key biological processes.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Pseudomonas aeruginosa is a multi-drug resistant, human opportunistic pathogen. If left untreated, P. aeruginosa can cause severe to life-threatening infections in people with burns, cystic fibrosis, and in immunocompromised patients. During chronic infections, P. aeruginosa primarily co-ordinates virulence in the host through a cell-to-cell communication mechanism called quorum sensing (QS). There are three key QS systems in P. aeruginosa responsible for driving global changes in virulence gene expression: the las, rhl, and pqs systems. Each of the las, rhl, and pqs systems rely on a receptor-autoinducer relationship: these receptor-autoinducer complexes are LasR-OdDHL, RhlR-BHL, and PqsR-PQS, respectively. When the receptors (LasR, RhlR, PqsR) bind with their cognate autoinducer (OdDHL, BHL, PQS, respectively), they act as transcription factors that ultimately stimulate the expression of hundreds of virulence-associated genes. The influence these QS systems have on the expression of virulence determinants has led to decades of scientific research focusing on the characterisation of these regulators. Although LasR and PqsR have been structurally elucidated, the RhlR crystal structure has long eluded characterisation and has been highly sought after due to its obvious potential as a therapeutic target.
In a collaborative research effort, I helped to identify ten additional proteins as putative binding partners of the pqs autoinducer, PQS. Four of the ten proteins identified were the cyanide synthase (HcnC), a putative protease (PfpI), a phenazine biosynthetic protein (PhzD1), and the QS regulator RhlR. For this PhD project, I aimed to structurally and biochemically characterise these four proteins to, in part, confirm their proposed interaction with PQS. A novel ligand (benzoic acid) was discovered bound in the active site of PhzD1 (crystal structure solved to 1.1 Å). Additionally, the crystal structure for PfpI was resolved at 1.4 Å resolution. The PfpI tertiary and quaternary structures obtained in this study suggested a possible role in electrophile detoxification, a hypothesis which I confirmed in vitro using 1D NMR. To complement the novel PfpI structural and biochemical data, I generated and confirmed “clean” pfpI deletion mutants for phenotypic and ‘omic analyses. I observed discrepancies in phenotypes between the pfpI deletion mutant and the pfpI transposon mutants previously reported in the published literature, which I sought to reconcile through subsequent whole genome sequencing (WGS) of these previously published strains. WGS of the pfpI transposon mutants revealed a plethora of unexpected mutations elsewhere in the genome, which likely contribute to many of the reported phenotypes. The “clean” deletion mutant that I generated harboured no significant additional mutations. Proteomic profiling of the pfpI deletion mutant exhibited altered protein expression in systems involved in Type VI secretion, motility, and metabolism.
Overall, the work presented in this dissertation further illustrates the intractability of purifying the QS transcriptional regulator, RhlR. I report benzoic acid to be a novel binding partner for the phenazine biosynthetic protein, PhzD1. Phenotypic analyses of pfpI mutants and consequent WGS highlight the need for rigorous strain validation when using transposon mutant libraries. Using the PfpI structural data I obtained during this study, I hypothesised and confirmed a novel detoxification role for PfpI in P. aeruginosa. Lastly, proteomic analysis of a pfpI deficient mutant revealed global dysregulation of key biological processes. |
Tittes, Yves Ulrich The Architecture of Polyketide Synthases Journal Article In: Thesis/Dissertation, 2023. @article{noKey,
title = {The Architecture of Polyketide Synthases},
author = {Tittes, Yves Ulrich},
url = {https://edoc.unibas.ch/94195/1/Thesis_YT_edoc_wCC_a.pdf},
doi = {Thesis},
year = {2023},
date = {2023-02-20},
journal = {Thesis/Dissertation},
abstract = {Since the discovery of penicillin over a century ago, secondary metabolites from all kingdoms of life have proven to be of high medical value. One class of proteins prevalent in the production of secondary metabolites are polyketide synthases (PKSs). Their polyketide products are complex organic compounds based on carbon chains assembled from carboxylic acid precursors. Many polyketides are produced by their hosts with the primary purpose of gaining an advantage in their ecological niche. To contribute to such an advantage, a significant proportion of polyketides are active against pro- and eukaryotic microorganisms. Type I PKSs are giant multienzyme proteins employing an assembly line logic for the synthesis of the most complex polyketides. They are composed of one or more functional and structural modules, each capable of carrying out one step of precursor elongation during the formation of an extended polyketide product.
In this thesis, I address two fundamental and open questions in the biosynthesis of polyketides: First, what is the unique architecture underlying the assembly line logic of multimodular PKS assembly lines; and second, how is atomic accuracy achieved in cyclization and aromatic ring formation in the final step of PKS action.
The first aim is addressed in chapter two, which provides for the first time detailed structural insights into the organization of type I PKS multimodules. This is achieved by cryo-electron microscopic analysis of filamentous and non-filamentous forms of K3DAK4, a bimodular trans-acyltransferase (AT) PKS fragment from Brevibacillus brevis. Overall reconstructions are provided at an intermediate resolution of 7 Å, with detailed insights into individual domains at sub-3Å resolution from cryo-electron microscopy and X-ray crystallography. The bimodule core displays a vertical stacking of its two modules along the central dimer axis of all three enzymatic domains involved. Additionally, K3DAK4 oligomerizes into filaments horizontally via small scaffolding domains in a trans-AT PKS-specific manner.
In chapter three the second aim is tackled, as I visualize an intermediate of the enigmatic targeted cyclization and aromatic ring formation in the product template domain (PT) of the aflatoxin-producing PksA at 2.7 Å resolution using X-ray crystallography. To this end a substrate-analogue mimicking the transient intermediate after the first of two cyclization steps facilitated by the enzyme is covalently crosslinked to the active site. The positioning of the ligand relative to previously known ligands representing the pre-and post-cyclization states indicate an outward movement of the substrate throughout the process and a substantial effect of progressing cyclization on the meticulous positioning of the intermediates.
The work provides detailed insights into core aspects of PKS biology from the atomistic picture of guided product modification to the giant overall assembly line architecture. In chapter four, both of these levels are put into context with current advances in the analysis of modular structure and dynamics of PKSs, such as recent structural models of cis-AT PKS modules and iterative PKSs. Furthermore, it addresses currently open questions, such as the interaction of trans-AT PKS with their cognate trans-acting enzymes. Altogether, the current progress in mechanistic understanding of PKS systems makes systematic and structure-guided efforts to unleash the full potential of PKS bioengineering ever more achievable.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Since the discovery of penicillin over a century ago, secondary metabolites from all kingdoms of life have proven to be of high medical value. One class of proteins prevalent in the production of secondary metabolites are polyketide synthases (PKSs). Their polyketide products are complex organic compounds based on carbon chains assembled from carboxylic acid precursors. Many polyketides are produced by their hosts with the primary purpose of gaining an advantage in their ecological niche. To contribute to such an advantage, a significant proportion of polyketides are active against pro- and eukaryotic microorganisms. Type I PKSs are giant multienzyme proteins employing an assembly line logic for the synthesis of the most complex polyketides. They are composed of one or more functional and structural modules, each capable of carrying out one step of precursor elongation during the formation of an extended polyketide product.
In this thesis, I address two fundamental and open questions in the biosynthesis of polyketides: First, what is the unique architecture underlying the assembly line logic of multimodular PKS assembly lines; and second, how is atomic accuracy achieved in cyclization and aromatic ring formation in the final step of PKS action.
The first aim is addressed in chapter two, which provides for the first time detailed structural insights into the organization of type I PKS multimodules. This is achieved by cryo-electron microscopic analysis of filamentous and non-filamentous forms of K3DAK4, a bimodular trans-acyltransferase (AT) PKS fragment from Brevibacillus brevis. Overall reconstructions are provided at an intermediate resolution of 7 Å, with detailed insights into individual domains at sub-3Å resolution from cryo-electron microscopy and X-ray crystallography. The bimodule core displays a vertical stacking of its two modules along the central dimer axis of all three enzymatic domains involved. Additionally, K3DAK4 oligomerizes into filaments horizontally via small scaffolding domains in a trans-AT PKS-specific manner.
In chapter three the second aim is tackled, as I visualize an intermediate of the enigmatic targeted cyclization and aromatic ring formation in the product template domain (PT) of the aflatoxin-producing PksA at 2.7 Å resolution using X-ray crystallography. To this end a substrate-analogue mimicking the transient intermediate after the first of two cyclization steps facilitated by the enzyme is covalently crosslinked to the active site. The positioning of the ligand relative to previously known ligands representing the pre-and post-cyclization states indicate an outward movement of the substrate throughout the process and a substantial effect of progressing cyclization on the meticulous positioning of the intermediates.
The work provides detailed insights into core aspects of PKS biology from the atomistic picture of guided product modification to the giant overall assembly line architecture. In chapter four, both of these levels are put into context with current advances in the analysis of modular structure and dynamics of PKSs, such as recent structural models of cis-AT PKS modules and iterative PKSs. Furthermore, it addresses currently open questions, such as the interaction of trans-AT PKS with their cognate trans-acting enzymes. Altogether, the current progress in mechanistic understanding of PKS systems makes systematic and structure-guided efforts to unleash the full potential of PKS bioengineering ever more achievable. |
Voss, Dr. Moritz Enzyme Engineering Enables Inversion of Substrate Stereopreference of the Halogenase WelO5* Journal Article In: The European Society Journal for Catalysis, 2022. @article{noKey,
title = {Enzyme Engineering Enables Inversion of Substrate Stereopreference of the Halogenase WelO5*},
author = {Voss, Dr. Moritz},
url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cctc.202201115},
doi = {https://doi.org/10.1002/cctc.202201115},
year = {2022},
date = {2022-12-20},
journal = {The European Society Journal for Catalysis},
abstract = {Enzymatic late-stage diversification of small molecules has the potential to rapidly generate diversity in compound libraries dedicated to drug discovery. In this context, freestanding Fe(II)/α-ketoglutarate-dependent halogenases have raised particular interest as this enzyme family allows the otherwise difficult regio- and stereoselective halogenation of unactivated C(sp3)−H bonds. Here, we report the development of two engineered variants of the halogenase WelO5* for the racemic resolution of a mixture of stereoisomers generated in the synthesis of a bioactive martinelline-derived fragment. By screening a 3-site combinatorial variant library, we could identify two variants exhibiting exquisite substrate selectivity towards the desired enantiomers. Strikingly, the inversion of substrate stereopreference between the halogenase variants was achieved by varying only three residues in the active site. Protein crystallization and subsequent structure elucidation of the wildtype enzyme and a WelO5* variant shed light on the factors governing substrate acceptance and selectivity.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Enzymatic late-stage diversification of small molecules has the potential to rapidly generate diversity in compound libraries dedicated to drug discovery. In this context, freestanding Fe(II)/α-ketoglutarate-dependent halogenases have raised particular interest as this enzyme family allows the otherwise difficult regio- and stereoselective halogenation of unactivated C(sp3)−H bonds. Here, we report the development of two engineered variants of the halogenase WelO5* for the racemic resolution of a mixture of stereoisomers generated in the synthesis of a bioactive martinelline-derived fragment. By screening a 3-site combinatorial variant library, we could identify two variants exhibiting exquisite substrate selectivity towards the desired enantiomers. Strikingly, the inversion of substrate stereopreference between the halogenase variants was achieved by varying only three residues in the active site. Protein crystallization and subsequent structure elucidation of the wildtype enzyme and a WelO5* variant shed light on the factors governing substrate acceptance and selectivity. |
Voss, Dr. Moritz, Hüppi, Sean Enzyme engineering enables inversion of substrate stereopreference of the halogenase WelO5 Journal Article In: Chemistry Europe, 2022. @article{noKey,
title = {Enzyme engineering enables inversion of substrate stereopreference of the halogenase WelO5},
author = {Voss, Dr. Moritz, Hüppi, Sean},
url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cctc.202201115},
doi = {https://doi.org/10.1002/cctc.202201115},
year = {2022},
date = {2022-10-23},
journal = {Chemistry Europe},
abstract = {Enzymatic late-stage diversification of small molecules has the potential to rapidly generate diversity in compound libraries dedicated to drug discovery. In this context, freestanding Fe(II)/α-ketoglutarate-dependent halogenases have raised particular interest as this enzyme family allows the otherwise difficult regio- and stereoselective halogenation of unactivated C(sp3)−H bonds. Here, we report the development of two engineered variants of the halogenase WelO5* for the racemic resolution of a mixture of stereoisomers generated in the synthesis of a bioactive martinelline-derived fragment. By screening a 3-site combinatorial variant library, we could identify two variants exhibiting exquisite substrate selectivity towards the desired enantiomers. Strikingly, the inversion of substrate stereopreference between the halogenase variants was achieved by varying only three residues in the active site. Protein crystallization and subsequent structure elucidation of the wildtype enzyme and a WelO5* variant shed light on the factors governing substrate acceptance and selectivity.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Enzymatic late-stage diversification of small molecules has the potential to rapidly generate diversity in compound libraries dedicated to drug discovery. In this context, freestanding Fe(II)/α-ketoglutarate-dependent halogenases have raised particular interest as this enzyme family allows the otherwise difficult regio- and stereoselective halogenation of unactivated C(sp3)−H bonds. Here, we report the development of two engineered variants of the halogenase WelO5* for the racemic resolution of a mixture of stereoisomers generated in the synthesis of a bioactive martinelline-derived fragment. By screening a 3-site combinatorial variant library, we could identify two variants exhibiting exquisite substrate selectivity towards the desired enantiomers. Strikingly, the inversion of substrate stereopreference between the halogenase variants was achieved by varying only three residues in the active site. Protein crystallization and subsequent structure elucidation of the wildtype enzyme and a WelO5* variant shed light on the factors governing substrate acceptance and selectivity. |
Sheehan, Colin T., Hampton, Thomas H. Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10 Journal Article In: Journal of Biological Chemistry, 2022. @article{noKey,
title = {Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10},
author = {Sheehan, Colin T., Hampton, Thomas H.},
url = {https://www.sciencedirect.com/science/article/pii/S0021925822009966},
doi = {https://doi.org/10.1016/j.jbc.2022.102552},
year = {2022},
date = {2022-09-28},
journal = {Journal of Biological Chemistry},
abstract = {Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of these G3BP1 affinities, and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that co-purification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of these G3BP1 affinities, and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that co-purification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes. |
Dolton, Garry, Rius, Cristina Emergence of immune escape at dominant SARSCoV- 2 killer T cell epitope Journal Article In: Cell Press, 2022. @article{noKey,
title = {Emergence of immune escape at dominant SARSCoV- 2 killer T cell epitope},
author = {Dolton, Garry, Rius, Cristina},
url = {https://pubmed.ncbi.nlm.nih.gov/35931021/},
doi = {https://doi.org/10.1016/j.cell.2022.07.002},
year = {2022},
date = {2022-08-03},
journal = {Cell Press},
abstract = {We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent
human leukocyte antigen (HLA) class I worldwide, HLA A*02. The Spike P272L mutation that has arisen in at
least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘‘variants of concern,’’
was not recognized by the large CD8 T cell response seen across cohorts of HLA A*02+ convalescent patients
and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different
individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may
be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike,
providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting
the need for monitoring T cell escape in new SARS-CoV-2 variants.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent
human leukocyte antigen (HLA) class I worldwide, HLA A*02. The Spike P272L mutation that has arisen in at
least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘‘variants of concern,’’
was not recognized by the large CD8 T cell response seen across cohorts of HLA A*02+ convalescent patients
and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different
individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may
be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike,
providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting
the need for monitoring T cell escape in new SARS-CoV-2 variants. |
Tatsis, Polychronis The iron-entry pores in Dps-like proteins as a potential drug-design target: Structural investigations Journal Article In: Thesis/Dissertation, 2022. @article{noKey,
title = {The iron-entry pores in Dps-like proteins as a potential drug-design target: Structural investigations},
author = {Tatsis, Polychronis},
url = {https://www.utupub.fi/bitstream/handle/10024/154499/Tatsis_Polychronis_Thesis.pdf?sequence=1},
doi = {Thesis},
year = {2022},
date = {2022-05-29},
journal = {Thesis/Dissertation},
abstract = {Streptococcus suis is an emerging catalase-negative zoonotic pathogen that uses the peroxide resistance protein (Dpr) as a defensive mechanism against oxidative stress. Dpr belongs to a family of proteins that form spherical dodecamers with a hollow cavity in the middle. Dpr, as other members of the family, uses four pores found on the surface of the dodecamer and formed by the N-terminals of adjacent monomers (N-terminal pores) to take up Fe2+ and deposit it inside the cavity after its oxidation to Fe3+ in ferroxidase sites in the interior of the dodecamer. In this way, the generation of toxic hydroxyl radicals via Fenton’s reaction is avoided. In this study, a new purification process and crystallization conditions for Dpr were found. Besides, the ligandability of Dpr for use as a drug target was investigated. 6xHis-tagged Dpr was successfully produced and purified. Crystallization screens yielded crystals in 10 conditions and further optimization led to crystals suitable for structural analysis. Synchrotron X-ray data were collected to 2.2 Å resolution. A novel ligand library design led to an initial library of 82 compounds that could act as possible N-terminal pore blockers. After a score threshold of -7, twenty (20) ligands remained. Similar, to the latter ones, marketed ligands were retrieved, and ten (10) of them were kept, all sharing the feature of having aromatic rings. Phe133 was found as the only residue responsible for Pi-pi interactions with the ligands. This is the first successful approach for 6xHis-tag Dpr crystal production and structure determination. It is also the first approach for ligand creation against the N-terminal pores of the Dpr, setting the basis for new possible future therapeutic approaches for S. suis-related infections treatment, avoiding the obstacle of antibiotic resistance.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Streptococcus suis is an emerging catalase-negative zoonotic pathogen that uses the peroxide resistance protein (Dpr) as a defensive mechanism against oxidative stress. Dpr belongs to a family of proteins that form spherical dodecamers with a hollow cavity in the middle. Dpr, as other members of the family, uses four pores found on the surface of the dodecamer and formed by the N-terminals of adjacent monomers (N-terminal pores) to take up Fe2+ and deposit it inside the cavity after its oxidation to Fe3+ in ferroxidase sites in the interior of the dodecamer. In this way, the generation of toxic hydroxyl radicals via Fenton’s reaction is avoided. In this study, a new purification process and crystallization conditions for Dpr were found. Besides, the ligandability of Dpr for use as a drug target was investigated. 6xHis-tagged Dpr was successfully produced and purified. Crystallization screens yielded crystals in 10 conditions and further optimization led to crystals suitable for structural analysis. Synchrotron X-ray data were collected to 2.2 Å resolution. A novel ligand library design led to an initial library of 82 compounds that could act as possible N-terminal pore blockers. After a score threshold of -7, twenty (20) ligands remained. Similar, to the latter ones, marketed ligands were retrieved, and ten (10) of them were kept, all sharing the feature of having aromatic rings. Phe133 was found as the only residue responsible for Pi-pi interactions with the ligands. This is the first successful approach for 6xHis-tag Dpr crystal production and structure determination. It is also the first approach for ligand creation against the N-terminal pores of the Dpr, setting the basis for new possible future therapeutic approaches for S. suis-related infections treatment, avoiding the obstacle of antibiotic resistance. |
Sheehan, Colin T., Madden, Dean R. Stereochemistry of transient protein-protein interactions in a signaling hub: exploring G3BP1-mediated regulation of CFTR deubiquitination Journal Article In: The Journal of Biological Chemistry, 2022. @article{noKey,
title = {Stereochemistry of transient protein-protein interactions in a signaling hub: exploring G3BP1-mediated regulation of CFTR deubiquitination},
author = {Sheehan, Colin T., Madden, Dean R.},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9723946/},
doi = {https://doi.org/10.1016%2Fj.jbc.2022.102552},
year = {2022},
date = {2022-03-01},
journal = {The Journal of Biological Chemistry},
abstract = {Intrinsically disordered proteins (IDPs) can coordinate often transient or weak interactions with multiple proteins to mediate complex signals within large, reversible protein networks. Among these, the IDP hub protein G3BP1 forms protein complexes with Caprin1 and USP10, and the resulting control of USP10 activity plays an important role in a pathogenic virulence system that targets CFTR endocytic recycling. However, while the identities of protein interactors are known for many of these IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of the G3BP1 affinities, and show that substitution of G3BP1 residues F15 or F33 to tryptophan reduces affinity for both the USP10 and Caprin1 motif peptides. These same mutations significantly reduce formation of complexes by the full-length proteins. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with the USP10 and Caprin1 peptides. An amino-acid scan of the USP10 and Caprin1 motif peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific mutations with the potential to create higher affinity peptides. Taken together, these data show that small changes in 1:1 binding affinity can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Intrinsically disordered proteins (IDPs) can coordinate often transient or weak interactions with multiple proteins to mediate complex signals within large, reversible protein networks. Among these, the IDP hub protein G3BP1 forms protein complexes with Caprin1 and USP10, and the resulting control of USP10 activity plays an important role in a pathogenic virulence system that targets CFTR endocytic recycling. However, while the identities of protein interactors are known for many of these IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of the G3BP1 affinities, and show that substitution of G3BP1 residues F15 or F33 to tryptophan reduces affinity for both the USP10 and Caprin1 motif peptides. These same mutations significantly reduce formation of complexes by the full-length proteins. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with the USP10 and Caprin1 peptides. An amino-acid scan of the USP10 and Caprin1 motif peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific mutations with the potential to create higher affinity peptides. Taken together, these data show that small changes in 1:1 binding affinity can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes. |
Tan, Yaw Bia, Lello, Laura Sandra Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold Journal Article In: Nucleic Acid Research, 2022. @article{noKey,
title = {Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold},
author = {Tan, Yaw Bia, Lello, Laura Sandra},
url = {https://academic.oup.com/nar/article/50/2/1000/6509088},
doi = {https://doi.org/10.1093/nar/gkab1302},
year = {2022},
date = {2022-01-16},
journal = {Nucleic Acid Research},
abstract = {Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication. |
Sheehan, Colin T. New Perspectives on a Pseudomonas aeruginosa Virulence Pathway Affecting Ubiquitination and Protein Homeostasis Journal Article In: Thesis/Dissertation, 2021. @article{noKey,
title = {New Perspectives on a Pseudomonas aeruginosa Virulence Pathway Affecting Ubiquitination and Protein Homeostasis},
author = {Sheehan, Colin T.},
url = {https://www.proquest.com/openview/27dea45adf0131f73a5bed7e68f2957c/1?pq-origsite=gscholar&cbl=18750&diss=y},
doi = {Thesis},
year = {2021},
date = {2021-12-30},
journal = {Thesis/Dissertation},
abstract = {Protein homeostasis (proteostasis) refers to the dynamic regulation of a
stable and functional proteome. The extensive proteostasis network includes
integrated cellular mechanisms that control biogenesis, folding, trafficking, and
degradation of proteins. Posttranslational modification and protein degradation are
key pathways that minimize homeostatic perturbations; however, disease occurs
when these processes become dysregulated. Loss of protein homeostasis such
as by an increase in misfolded proteins contributes to the pathology of many
disorders including cancers to neurodegenerative diseases. Maintaining the
integrity of the proteome is essential for viability, but cells continuously face
extracellular and intracellular stresses that destabilize protein homeostasis.
Collaborations among several laboratories at Dartmouth led to the
discovery of virulence factor secreted from Pseudomonas aeruginosa that
dysregulates the homeostasis of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). The virulence factor, named the CFTR inhibitory
factor (Cif), decreases the number of CFTR channels on the plasma membrane by
manipulating the host ubiquitination system. Cif causes a complex to form between
G3BP1 and USP10, which renders USP10’s unable to perform its deubiquitinase
activity. Unfortunately, the mechanism of Cif-mediated G3BP1:USP10 complex
formation remains unknown. Cif causes protein dyshomeostasis resulting in
decreased CFTR level which ultimately aids bacterial colonization of the
compromised host.
The work detailed within expands our working model of Cif virulence to
include additional proteins and cellular phenomena. Caprin1, which was not included in previous investigations, competes with USP10 to bind G3BP1. We
provide a detailed stereochemical investigation of the G3BP1:USP10 and
G3BP1:Caprin1 complexes. Further, we describe discrete proteins mutations and
their effects on protein homeostasis. In parallel, we develop molecular tools to
determine the roles of G3BP1, USP10, and Caprin1 in the Cif virulence pathway.
These results offer new insights into the mechanism of Cif-mediated protein
dyshomeostasis.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Protein homeostasis (proteostasis) refers to the dynamic regulation of a
stable and functional proteome. The extensive proteostasis network includes
integrated cellular mechanisms that control biogenesis, folding, trafficking, and
degradation of proteins. Posttranslational modification and protein degradation are
key pathways that minimize homeostatic perturbations; however, disease occurs
when these processes become dysregulated. Loss of protein homeostasis such
as by an increase in misfolded proteins contributes to the pathology of many
disorders including cancers to neurodegenerative diseases. Maintaining the
integrity of the proteome is essential for viability, but cells continuously face
extracellular and intracellular stresses that destabilize protein homeostasis.
Collaborations among several laboratories at Dartmouth led to the
discovery of virulence factor secreted from Pseudomonas aeruginosa that
dysregulates the homeostasis of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). The virulence factor, named the CFTR inhibitory
factor (Cif), decreases the number of CFTR channels on the plasma membrane by
manipulating the host ubiquitination system. Cif causes a complex to form between
G3BP1 and USP10, which renders USP10’s unable to perform its deubiquitinase
activity. Unfortunately, the mechanism of Cif-mediated G3BP1:USP10 complex
formation remains unknown. Cif causes protein dyshomeostasis resulting in
decreased CFTR level which ultimately aids bacterial colonization of the
compromised host.
The work detailed within expands our working model of Cif virulence to
include additional proteins and cellular phenomena. Caprin1, which was not included in previous investigations, competes with USP10 to bind G3BP1. We
provide a detailed stereochemical investigation of the G3BP1:USP10 and
G3BP1:Caprin1 complexes. Further, we describe discrete proteins mutations and
their effects on protein homeostasis. In parallel, we develop molecular tools to
determine the roles of G3BP1, USP10, and Caprin1 in the Cif virulence pathway.
These results offer new insights into the mechanism of Cif-mediated protein
dyshomeostasis. |
Gundesø, Sigurd Eidem Structural and functional studies of Ectoine Synthase from Chromohalobacter salexigens DSM 3043 and Marinobacter sp. CK1 Journal Article In: Thesis/Dissertation, 2021. @article{noKey,
title = {Structural and functional studies of Ectoine Synthase from Chromohalobacter salexigens DSM 3043 and Marinobacter sp. CK1},
author = {Gundesø, Sigurd Eidem},
url = {https://munin.uit.no/handle/10037/26585},
doi = {Thesis},
year = {2021},
date = {2021-10-14},
journal = {Thesis/Dissertation},
abstract = {Ectoine is a compatible solute found in many microorganisms adapted to survive in saline and other extreme environments. Here, it aids microorganisms to counter osmotic stress and protect their enzymes. Ectoine exhibit many interesting properties that is potentially commercially exploitable, and it is currently produced and found in several products on the market. While ectoine is produced by whole cell synthesis, the EctABC enzymes in the biosynthesis pathway of ectoine was currently not well described structurally or functionally. Here, we present structural and biochemical characterizations of ectoine synthase from two organisms, Chromohalobacter salexigens DSM3043 and Marinobacter sp. CK1. We cloned, expressed and expression optimized both candidates, and purified them by immobilized metal affinity chromatography and gel filtration. C. salexigens EctC (CSEctC) yielded 14-18 mg/L cell culture while Marinobacter sp. CK1 (MarEctC) yielded 0.75-1.5 mg/L culture. We then produced diffracting crystals of CSEctC and obtained a data set from which the structure of CSEctC was determined. We further obtained preliminary biochemical data relating to thermostability and activity from both candidates. The crystal structure from CSEctC shows that it is adapts a typical β-sandwich fold, consistent with earlier structural investigations of other EctC type proteins. This study provides a solid foundation for further research on EctC from our model organisms, and protocols and techniques developed herein can be further optimized to obtain more biochemical data about this interesting enzyme.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Ectoine is a compatible solute found in many microorganisms adapted to survive in saline and other extreme environments. Here, it aids microorganisms to counter osmotic stress and protect their enzymes. Ectoine exhibit many interesting properties that is potentially commercially exploitable, and it is currently produced and found in several products on the market. While ectoine is produced by whole cell synthesis, the EctABC enzymes in the biosynthesis pathway of ectoine was currently not well described structurally or functionally. Here, we present structural and biochemical characterizations of ectoine synthase from two organisms, Chromohalobacter salexigens DSM3043 and Marinobacter sp. CK1. We cloned, expressed and expression optimized both candidates, and purified them by immobilized metal affinity chromatography and gel filtration. C. salexigens EctC (CSEctC) yielded 14-18 mg/L cell culture while Marinobacter sp. CK1 (MarEctC) yielded 0.75-1.5 mg/L culture. We then produced diffracting crystals of CSEctC and obtained a data set from which the structure of CSEctC was determined. We further obtained preliminary biochemical data relating to thermostability and activity from both candidates. The crystal structure from CSEctC shows that it is adapts a typical β-sandwich fold, consistent with earlier structural investigations of other EctC type proteins. This study provides a solid foundation for further research on EctC from our model organisms, and protocols and techniques developed herein can be further optimized to obtain more biochemical data about this interesting enzyme. |
Rangel, Victor Lopes X-ray crystallographic fragment screening against the human prion protein Journal Article In: Thesis/Dissertation, 2021. @article{noKey,
title = {X-ray crystallographic fragment screening against the human prion protein},
author = {Rangel, Victor Lopes},
url = {https://www.teses.usp.br/teses/disponiveis/60/60136/tde-27092021-150052/pt-br.php},
doi = {https://doi.org/10.11606/T.60.2021.tde-27092021-150052},
year = {2021},
date = {2021-09-27},
journal = {Thesis/Dissertation},
abstract = {Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an "undruggable" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an "undruggable" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors. |
T. Holleman, Ethan Polo: an open-source graphical user interface for crystallization screening Journal Article In: Journal of Applied Crystallography, 2021. @article{noKey,
title = {Polo: an open-source graphical user interface for crystallization screening},
author = {T. Holleman, Ethan},
url = {https://onlinelibrary.wiley.com/iucr/doi/10.1107/S1600576721000108},
doi = {https://doi.org/10.1107/S1600576721000108},
year = {2021},
date = {2021-03-31},
journal = {Journal of Applied Crystallography},
abstract = {Polo is a Python-based graphical user interface designed to streamline viewing and analysis of images to monitor crystal growth, with a specific target to enable users of the High-Throughput Crystallization Screening Center at Hauptman-Woodward Medical Research Institute (HWI) to efficiently inspect their crystallization experiments. Polo aims to increase efficiency, reducing time spent manually reviewing crystallization images, and to improve the potential of identifying positive crystallization conditions. Polo provides a streamlined one-click graphical interface for the Machine Recognition of Crystallization Outcomes (MARCO) convolutional neural network for automated image classification, as well as powerful tools to view and score crystallization images, to compare crystallization conditions, and to facilitate collaborative review of crystallization screening results. Crystallization images need not have been captured at HWI to utilize Polo's basic functionality. Polo is free to use and modify for both academic and commercial use under the terms of the copyleft GNU General Public License v3.0.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
Polo is a Python-based graphical user interface designed to streamline viewing and analysis of images to monitor crystal growth, with a specific target to enable users of the High-Throughput Crystallization Screening Center at Hauptman-Woodward Medical Research Institute (HWI) to efficiently inspect their crystallization experiments. Polo aims to increase efficiency, reducing time spent manually reviewing crystallization images, and to improve the potential of identifying positive crystallization conditions. Polo provides a streamlined one-click graphical interface for the Machine Recognition of Crystallization Outcomes (MARCO) convolutional neural network for automated image classification, as well as powerful tools to view and score crystallization images, to compare crystallization conditions, and to facilitate collaborative review of crystallization screening results. Crystallization images need not have been captured at HWI to utilize Polo's basic functionality. Polo is free to use and modify for both academic and commercial use under the terms of the copyleft GNU General Public License v3.0. |
Mieczkowski, Mateusz Structural investigation of functional nucleic acids Journal Article In: Thesis/Dissertation, 2021. @article{noKey,
title = {Structural investigation of functional nucleic acids},
author = {Mieczkowski, Mateusz},
url = {https://ediss.uni-goettingen.de/handle/21.11130/00-1735-0000-0008-5885-F},
doi = {Thesis},
year = {2021},
date = {2021-03-25},
journal = {Thesis/Dissertation},
abstract = {DNA enzymes, also known as deoxyribozymes, are synthetic single-stranded DNA molecules able
to catalyze chemical reactions. There are two main reasons for studying deoxyribozymes: their
practical value in various applications, and the understanding of basic properties - such as folding
and catalysis - of a biopolymer that is of central importance for life. Compared to ribozymes, the
DNA enzymes have a potential value as tools for industrial or therapeutic applications, owing to
more cost-effective synthesis and higher stability. The first crystal structure of a deoxyribozyme
demonstrated that DNA possesses the intrinsic ability to adopt complex tertiary folds that support
catalysis and unveiled the active site of a DNA enzyme in the post-catalytic state (Ponce-Salvatierra,
Wawrzyniak-Turek et al. 2016). The second reported crystal structure of the RNA-cleaving
deoxyribozyme complements observations about the folds and catalysis of DNA enzymes although
the structure was derived with DNA as a substrate mimic of RNA (Liu, Yu et al. 2017). These
crystal structures represent a breakthrough in the field, but they are still insufficient to derive a clear
mechanistic picture of the specific features of different RNA ligating and RNA cleaving
deoxyribozymes. Therefore, ongoing efforts are devoted to structurally investigating additional
deoxyribozymes. The new DNA enzymes were evolved to discriminate modified and unmodified
RNA substrates and provide attractive tools for studying the natural epitranscriptomic RNA
modification N6-methyladenosine (Sednev, Mykhailiuk et al. 2018). In the present study, the goal
is to elucidate the structural basis for recognition of the methylated nucleobase by solving the
crystal structure of the m6A sensitive RNA-cleaving deoxyribozyme in complex with an
uncleavable analog of the RNA substrate, containing either methylated and unmethylated
adenosine. Surprisingly, the RNA substrate dissociated from the deoxyribozyme during the
crystallization process. Two structures for unmethylated and one of the methylated RNA substrate
analog were solved. The next goal is to elucidate the crystal structure of the RNA-ligating
deoxyribozyme in the pre-catalytic state of reaction. The previously reported crystal structure of
the 9DB1 in the post-catalytic state of reaction could not explain the role of magnesium cations as
cofactors for accelerating RNA ligation and properly describe the ligation mechanism. The
structural investigation of the 9DB1 in the pre-catalytic state resulted in the ligation of the two
RNA substrates during the crystallization process. In the future, other strategies are necessary to
solve the questions on substrate recognition and catalytic mechanism of the RNA-cleaving and
RNA-ligating deoxyribozymes investigated in this study.
The second project deals with synthetic RNA aptamers that were identified by in vitro selection to
mimic fluorescent proteins for RNA imaging and the development of biosensors. Several examples
2
of fluorogen-activating RNA aptamers are known, and for some, the crystal structures have
recently been solved e.g. of the Spinach, Mango, and Corn aptamers, that bind synthetic analogs
of the GFP chromophore (Neubacher and Hennig 2019). The Chili is a new fluorogenic-RNA
aptamer that mimics large Stokes shift (LSS) fluorescent proteins (FPs) by inducing highly Stokesshifted
emission from several new green and red HBI (4-hydroxybenzylidene imidazolinone)
derivatives that are non‐fluorescent when free in solution (Steinmetzger, Palanisamy et al. 2019).
The new fluorophores are the first variants of fluorogenic aptamer ligands with permanently
cationic sidechains that are bound by the RNA in their protonated phenol form, while emission
occurs from the phenolate intermediate after excited-state proton transfer. The Chili–DMHBO+
complex is the longest wavelength-emitting (592 nm) and tightest binding (KD=12 nM) RNA
fluorophore currently known in the growing family of HBI-binding aptamers. By employing X-ray
crystallography, I have elucidated the three-dimensional structure of the Chili fluorophore binding
site and revealed the structural basis for the large apparent Stokes shift and the promiscuity of the
Chili aptamer to activate red and green-emitting chromophores.},
keywords = {Rock Maker®},
pubstate = {published},
tppubtype = {article}
}
DNA enzymes, also known as deoxyribozymes, are synthetic single-stranded DNA molecules able
to catalyze chemical reactions. There are two main reasons for studying deoxyribozymes: their
practical value in various applications, and the understanding of basic properties - such as folding
and catalysis - of a biopolymer that is of central importance for life. Compared to ribozymes, the
DNA enzymes have a potential value as tools for industrial or therapeutic applications, owing to
more cost-effective synthesis and higher stability. The first crystal structure of a deoxyribozyme
demonstrated that DNA possesses the intrinsic ability to adopt complex tertiary folds that support
catalysis and unveiled the active site of a DNA enzyme in the post-catalytic state (Ponce-Salvatierra,
Wawrzyniak-Turek et al. 2016). The second reported crystal structure of the RNA-cleaving
deoxyribozyme complements observations about the folds and catalysis of DNA enzymes although
the structure was derived with DNA as a substrate mimic of RNA (Liu, Yu et al. 2017). These
crystal structures represent a breakthrough in the field, but they are still insufficient to derive a clear
mechanistic picture of the specific features of different RNA ligating and RNA cleaving
deoxyribozymes. Therefore, ongoing efforts are devoted to structurally investigating additional
deoxyribozymes. The new DNA enzymes were evolved to discriminate modified and unmodified
RNA substrates and provide attractive tools for studying the natural epitranscriptomic RNA
modification N6-methyladenosine (Sednev, Mykhailiuk et al. 2018). In the present study, the goal
is to elucidate the structural basis for recognition of the methylated nucleobase by solving the
crystal structure of the m6A sensitive RNA-cleaving deoxyribozyme in complex with an
uncleavable analog of the RNA substrate, containing either methylated and unmethylated
adenosine. Surprisingly, the RNA substrate dissociated from the deoxyribozyme during the
crystallization process. Two structures for unmethylated and one of the methylated RNA substrate
analog were solved. The next goal is to elucidate the crystal structure of the RNA-ligating
deoxyribozyme in the pre-catalytic state of reaction. The previously reported crystal structure of
the 9DB1 in the post-catalytic state of reaction could not explain the role of magnesium cations as
cofactors for accelerating RNA ligation and properly describe the ligation mechanism. The
structural investigation of the 9DB1 in the pre-catalytic state resulted in the ligation of the two
RNA substrates during the crystallization process. In the future, other strategies are necessary to
solve the questions on substrate recognition and catalytic mechanism of the RNA-cleaving and
RNA-ligating deoxyribozymes investigated in this study.
The second project deals with synthetic RNA aptamers that were identified by in vitro selection to
mimic fluorescent proteins for RNA imaging and the development of biosensors. Several examples
2
of fluorogen-activating RNA aptamers are known, and for some, the crystal structures have
recently been solved e.g. of the Spinach, Mango, and Corn aptamers, that bind synthetic analogs
of the GFP chromophore (Neubacher and Hennig 2019). The Chili is a new fluorogenic-RNA
aptamer that mimics large Stokes shift (LSS) fluorescent proteins (FPs) by inducing highly Stokesshifted
emission from several new green and red HBI (4-hydroxybenzylidene imidazolinone)
derivatives that are non‐fluorescent when free in solution (Steinmetzger, Palanisamy et al. 2019).
The new fluorophores are the first variants of fluorogenic aptamer ligands with permanently
cationic sidechains that are bound by the RNA in their protonated phenol form, while emission
occurs from the phenolate intermediate after excited-state proton transfer. The Chili–DMHBO+
complex is the longest wavelength-emitting (592 nm) and tightest binding (KD=12 nM) RNA
fluorophore currently known in the growing family of HBI-binding aptamers. By employing X-ray
crystallography, I have elucidated the three-dimensional structure of the Chili fluorophore binding
site and revealed the structural basis for the large apparent Stokes shift and the promiscuity of the
Chili aptamer to activate red and green-emitting chromophores. |
