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Article ; Online: Evolving SAXS versatility: solution X-ray scattering for macromolecular architecture, functional landscapes, and integrative structural biology.

Brosey, Chris A / Tainer, John A

2019 Volume 58, Page(s) 197–213

Abstract: Small-angle X-ray scattering (SAXS) has emerged as an enabling integrative technique for comprehensive analyses of macromolecular structures and interactions in solution. Over the past two decades, SAXS has become a mainstay of the structural biologist's ...

Abstract	Small-angle X-ray scattering (SAXS) has emerged as an enabling integrative technique for comprehensive analyses of macromolecular structures and interactions in solution. Over the past two decades, SAXS has become a mainstay of the structural biologist's toolbox, supplying multiplexed measurements of molecular shape and dynamics that unveil biological function. Here, we discuss evolving SAXS theory, methods, and applications that extend the field of small-angle scattering beyond simple shape characterization. SAXS, coupled with size-exclusion chromatography (SEC-SAXS) and time-resolved (TR-SAXS) methods, is now providing high-resolution insight into macromolecular flexibility and ensembles, delineating biophysical landscapes, and facilitating high-throughput library screening to assess macromolecular properties and to create opportunities for drug discovery. Looking forward, we consider SAXS in the integrative era of hybrid structural biology methods, its potential for illuminating cellular supramolecular and mesoscale structures, and its capacity to complement high-throughput bioinformatics sequencing data. As advances in the field continue, we look forward to proliferating uses of SAXS based upon its abilities to robustly produce mechanistic insights for biology and medicine.
MeSH term(s)	Macromolecular Substances/chemistry ; Macromolecular Substances/metabolism ; Scattering, Small Angle ; Solutions ; X-Ray Diffraction
Chemical Substances	Macromolecular Substances ; Solutions
Language	English
Publishing date	2019-06-13
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	1068353-7
ISSN	1879-033X ; 0959-440X
ISSN (online)	1879-033X
ISSN	0959-440X
DOI	10.1016/j.sbi.2019.04.004
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Article ; Online: Visualizing and accessing correlated SAXS data sets with Similarity Maps and Simple Scattering web resources.

Murray, Daniel T / Shin, David S / Classen, Scott / Brosey, Chris A / Hura, Greg L

Methods in enzymology

2022 Volume 678, Page(s) 411–440

Abstract: Constructing a comprehensive understanding of macromolecular behavior from a set of correlated small angle scattering (SAS) data is aided by tools that analyze all scattering curves together. SAS experiments on biological systems can be performed on ... ...

Abstract	Constructing a comprehensive understanding of macromolecular behavior from a set of correlated small angle scattering (SAS) data is aided by tools that analyze all scattering curves together. SAS experiments on biological systems can be performed on specimens that are more easily prepared, modified, and formatted relative to those of most other techniques. An X-ray SAS measurement (SAXS) can be performed in less than a milli-second in-line with treatment steps such as purification or exposure to modifiers. These capabilities are valuable since biological macromolecules (proteins, polynucleotides, lipids, and carbohydrates) change conformation or assembly under specific conditions that often define their biological role. Furthermore, mutation or post-translational modification change their behavior and provides an avenue to tailor their mechanics. Here, we describe tools to combine multiple correlated SAS measurements for analysis and review their application to biological systems. The SAXS Similarity Map (SSM) compares a set of scattering curves and quantifies the similarity between them for display as a color on a grid. Visualizing an entire correlated data set with SSMs helps identify patterns that reveal biological functions. The SSM analysis is available as a web-based tool at https://sibyls.als.lbl.gov/saxs-similarity/. To make data available and promote tool development, we have also deployed a repository of correlated SAS data sets called Simple Scattering (available at https://simplescattering.com). The correlated data sets used to demonstrate the SSM are available on the Simple Scattering website. We expect increased utilization of correlated SAS measurements to characterize the tightly controlled mechanistic properties of biological systems and fine-tune engineered macromolecules for nanotechnology-based applications.
MeSH term(s)	X-Ray Diffraction ; Scattering, Small Angle ; Proteins ; Molecular Conformation ; Macromolecular Substances
Chemical Substances	Proteins ; Macromolecular Substances
Language	English
Publishing date	2022-11-03
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural
ISSN	1557-7988
ISSN (online)	1557-7988
DOI	10.1016/bs.mie.2022.09.024
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Article ; Online: Applying HT-SAXS to chemical ligand screening.

Brosey, Chris A / Shen, Runze / Moiani, Davide / Jones, Darin E / Burnett, Kathryn / Hura, Greg L / Tainer, John A

Methods in enzymology

2022 Volume 678, Page(s) 331–350

Abstract: Chemical probes are invaluable tools for investigating essential biological processes. Understanding how small-molecule probes engage biomolecular conformations is critical to developing their functional selectivity. High-throughput solution X-ray ... ...

Abstract	Chemical probes are invaluable tools for investigating essential biological processes. Understanding how small-molecule probes engage biomolecular conformations is critical to developing their functional selectivity. High-throughput solution X-ray scattering is well-positioned to profile target-ligand complexes during probe development, bringing conformational insight and selection to traditional ligand binding assays. Access to high-quality synchrotron SAXS datasets and high-throughput data analysis now allows routine academic users to incorporate conformational information into small-molecule development pipelines. Here we describe a general approach for benchmarking and preparing HT-SAXS chemical screens from small fragment libraries. Using the allosteric oxidoreductase Apoptosis-Inducing Factor (AIF) as an exemplary system, we illustrate how HT-SAXS efficiently identifies an allosteric candidate among hits of a microscale thermophoresis ligand screen. We discuss considerations for pursuing HT-SAXS chemical screening with other systems of interest and reflect on advances to extend screening throughput and sensitivity.
MeSH term(s)	X-Ray Diffraction ; Ligands ; Scattering, Small Angle ; Oxidoreductases ; Synchrotrons
Chemical Substances	Ligands ; Oxidoreductases (EC 1.-)
Language	English
Publishing date	2022-11-26
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural
ISSN	1557-7988
ISSN (online)	1557-7988
DOI	10.1016/bs.mie.2022.09.022
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Article ; Online: An efficient chemical screening method for structure-based inhibitors to nucleic acid enzymes targeting the DNA repair-replication interface and SARS CoV-2.

Moiani, Davide / Link, Todd M / Brosey, Chris A / Katsonis, Panagiotis / Lichtarge, Olivier / Kim, Youngchang / Joachimiak, Andrzej / Ma, Zhijun / Kim, In-Kwon / Ahmed, Zamal / Jones, Darin E / Tsutakawa, Susan E / Tainer, John A

Methods in enzymology

2021 Volume 661, Page(s) 407–431

Abstract: We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the ... ...

Abstract	We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.
MeSH term(s)	COVID-19 ; DNA Repair ; Humans ; Molecular Docking Simulation ; Nucleic Acids ; SARS-CoV-2 ; Severe Acute Respiratory Syndrome
Chemical Substances	Nucleic Acids
Language	English
Publishing date	2021-09-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1557-7988
ISSN (online)	1557-7988
DOI	10.1016/bs.mie.2021.09.003
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Article ; Online: What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses.

Brosey, Chris A / Ahmed, Zamal / Lees-Miller, Susan P / Tainer, John A

Methods in enzymology

2017 Volume 592, Page(s) 417–455

Abstract: DNA damage outcomes depend upon the efficiency and fidelity of DNA damage responses (DDRs) for different cells and damage. As such, DDRs represent tightly regulated prototypical systems for linking nanoscale biomolecular structure and assembly to the ... ...

Abstract	DNA damage outcomes depend upon the efficiency and fidelity of DNA damage responses (DDRs) for different cells and damage. As such, DDRs represent tightly regulated prototypical systems for linking nanoscale biomolecular structure and assembly to the biology of genomic regulation and cell signaling. However, the dynamic and multifunctional nature of DDR assemblies can render elusive the correlation between the structures of DDR factors and specific biological disruptions to the DDR when these structures are altered. In this chapter, we discuss concepts and strategies for combining structural, biophysical, and imaging techniques to investigate DDR recognition and regulation, and thus bridge sequence-level structural biochemistry to quantitative biological outcomes visualized in cells. We focus on representative DDR responses from PARP/PARG/AIF damage signaling in DNA single-strand break repair and nonhomologous end joining complexes in double-strand break repair. Methods with exemplary experimental results are considered with a focus on strategies for probing flexibility, conformational changes, and assembly processes that shape a predictive understanding of DDR mechanisms in a cellular context. Integration of structural and imaging measurements promises to provide foundational knowledge to rationally control and optimize DNA damage outcomes for synthetic lethality and for immune activation with resulting insights for biology and cancer interventions.
MeSH term(s)	Animals ; Apoptosis Inducing Factor/chemistry ; Apoptosis Inducing Factor/metabolism ; Cryoelectron Microscopy/methods ; Crystallography, X-Ray/methods ; DNA/chemistry ; DNA/genetics ; DNA/metabolism ; DNA Damage ; DNA Repair ; Genomic Instability ; Glycoside Hydrolases/chemistry ; Glycoside Hydrolases/metabolism ; Humans ; Microscopy, Fluorescence/methods ; Models, Molecular ; Neoplasms/genetics ; Neoplasms/metabolism ; Optical Imaging/methods ; Poly(ADP-ribose) Polymerases/chemistry ; Poly(ADP-ribose) Polymerases/metabolism ; Signal Transduction
Chemical Substances	Apoptosis Inducing Factor ; DNA (9007-49-2) ; Poly(ADP-ribose) Polymerases (EC 2.4.2.30) ; Glycoside Hydrolases (EC 3.2.1.-)
Language	English
Publishing date	2017-05-29
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1557-7988 ; 0076-6879
ISSN (online)	1557-7988
ISSN	0076-6879
DOI	10.1016/bs.mie.2017.04.005
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Article ; Online: Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities.

Moiani, Davide / Ronato, Daryl A / Brosey, Chris A / Arvai, Andrew S / Syed, Aleem / Masson, Jean-Yves / Petricci, Elena / Tainer, John A

Methods in enzymology

2018 Volume 601, Page(s) 205–241

Abstract: For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures ... ...

Abstract	For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors. We maintain that evolutionarily related targets logically provide molecular avatars, where this Sanskrit term for descent includes ideas of functional relationships and of being a physical embodiment of the target's essential features without requiring high sequence identity. Appropriate biochemical and cell assays provide quantitative measurements, and for biomedical impacts, any inhibitor's activity should be validated in human cells. Specificity is effectively shown empirically by testing if mutations blocking target activity remove cellular inhibitor impact. We propose this approach to be superior to experiments testing for lack of cross-reactivity among possible related enzymes, which is a challenging negative experiment. As an exemplary avatar system for protein and DNA allosteric conformational controls, we focus here on developing separation-of-function inhibitors for meiotic recombination 11 nuclease activities. This was achieved not by targeting the active site but rather by geometrically impacting loop motifs analogously to ribosome antibiotics. These loops are neighboring the dimer interface and active site act in sculpting dsDNA and ssDNA into catalytically competent complexes. One of our design constraints is to preserve DNA substrate binding to geometrically block competing enzymes and pathways from the damaged site. We validate our allosteric approach to controlling outcomes in human cells by reversing the radiation sensitivity and genomic instability in BRCA mutant cells.
MeSH term(s)	Allosteric Regulation ; Amino Acid Sequence ; Drug Design ; Endonucleases/antagonists & inhibitors ; Endonucleases/metabolism ; Evolution, Molecular ; Exonucleases/antagonists & inhibitors ; Exonucleases/metabolism ; Humans ; MRE11 Homologue Protein/antagonists & inhibitors ; MRE11 Homologue Protein/genetics ; MRE11 Homologue Protein/metabolism ; Protein Conformation ; Sensitivity and Specificity ; Sequence Alignment ; Sequence Analysis, Protein
Chemical Substances	MRE11 protein, human ; Endonucleases (EC 3.1.-) ; Exonucleases (EC 3.1.-) ; MRE11 Homologue Protein (EC 3.1.-)
Language	English
Publishing date	2018-02-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1557-7988 ; 0076-6879
ISSN (online)	1557-7988
ISSN	0076-6879
DOI	10.1016/bs.mie.2017.11.030
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Article ; Online: A quantitative assay reveals ligand specificity of the DNA scaffold repair protein XRCC1 and efficient disassembly of complexes of XRCC1 and the poly(ADP-ribose) polymerase 1 by poly(ADP-ribose) glycohydrolase.

Kim, In-Kwon / Stegeman, Roderick A / Brosey, Chris A / Ellenberger, Tom

The Journal of biological chemistry

2014 Volume 290, Issue 6, Page(s) 3775–3783

Abstract: The posttranslational modification of proteins with poly(ADP-ribose) (PAR) regulates protein-protein interactions in DNA repair, gene expression, chromatin structure, and cell fate determination. The PAR polymerase PARP1 binds to damaged chromatin and ... ...

Abstract	The posttranslational modification of proteins with poly(ADP-ribose) (PAR) regulates protein-protein interactions in DNA repair, gene expression, chromatin structure, and cell fate determination. The PAR polymerase PARP1 binds to damaged chromatin and synthesizes PAR chains to signal DNA damage and recruit the DNA repair scaffold, XRCC1. Pharmacological blockade of PARP1 enzymatic activity impairs XRCC1-dependent repair of DNA damage and selectively kills cancer cells lacking other DNA repair functions. As such, PARP inhibitors are promising new therapies for repair-deficient tumors such as BRCA mutated breast cancers. Although the XRCC1-PARP1 complex is relevant to the proposed therapeutic mechanism of PARP inhibitors, the physical makeup and dynamics of this complex are not well characterized at the molecular level. Here we describe a fluorescence-based, real-time assay that quantitatively monitors interactions between PARylated PARP1 and XRCC1. Using this assay, we show that the PAR posttranslational modification by itself is a high affinity ligand for XRCC1, requiring a minimum chain length of 7 ADP-ribose units in the oligo(ADP-ribose) ligand for a stable interaction with XRCC1. This discrete binding interface enables the PAR glycohydrolase (PARG) to completely disassemble the PARP1-XRCC1 complex without assistance from a mono(ADP-ribose) glycohydrolase. Our quantitative, real-time assay of PAR-dependent protein-protein interactions and PAR turnover by PARG is an excellent tool for high-throughput screening to identify pharmacological modulators of PAR metabolism that may be useful therapeutic alternatives to PARP inhibitors.
MeSH term(s)	Adenosine Diphosphate Ribose/metabolism ; Binding Sites ; DNA-Binding Proteins/chemistry ; DNA-Binding Proteins/metabolism ; Glycoside Hydrolases/metabolism ; Humans ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases/chemistry ; Poly(ADP-ribose) Polymerases/metabolism ; Protein Binding ; Protein Processing, Post-Translational ; Substrate Specificity ; X-ray Repair Cross Complementing Protein 1
Chemical Substances	DNA-Binding Proteins ; X-ray Repair Cross Complementing Protein 1 ; XRCC1 protein, human ; Adenosine Diphosphate Ribose (20762-30-5) ; PARP1 protein, human (EC 2.4.2.30) ; Poly (ADP-Ribose) Polymerase-1 (EC 2.4.2.30) ; Poly(ADP-ribose) Polymerases (EC 2.4.2.30) ; Glycoside Hydrolases (EC 3.2.1.-) ; poly ADP-ribose glycohydrolase (EC 3.2.1.143)
Language	English
Publishing date	2014-12-04
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M114.624718
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Article ; Online: Sample preparation methods to analyze DNA-induced structural changes in replication protein A.

Brosey, Chris A / Tsutakawa, Susan E / Chazin, Walter J

Methods in molecular biology (Clifton, N.J.)

2012 Volume 922, Page(s) 101–122

Abstract: Propagation and maintenance of the cellular genome are among the most fundamental cellular processes, encompassing pathways associated with DNA replication, damage response, and repair. Replication Protein A (RPA), the primary single-stranded DNA-binding ...

Abstract	Propagation and maintenance of the cellular genome are among the most fundamental cellular processes, encompassing pathways associated with DNA replication, damage response, and repair. Replication Protein A (RPA), the primary single-stranded DNA-binding protein (SSB) in eukaryotes, serves to protect ssDNA generated during these events and to recruit and organize other DNA-processing factors requiring access to ssDNA substrates. RPA engages ssDNA in distinct, progressive binding modes, which are thought to correspond to different functional states of the protein during the course of DNA processing. Structural characterization of these unique complexes has remained challenging, however, as RPA is a multi-domain protein characterized by a flexible, modular organization. Biophysical approaches that are well suited to probing time-varying architectures, such as NMR and small-angle X-ray and neutron scattering (SAXS/SANS), when integrated with computational methods, can provide critical insights into the architectural changes associated with RPA's different DNA-binding modes. The success of these methods, however, is highly contingent upon the purity, homogeneity, and stability of the sample under study. Here we describe a basic protocol for characterizing and optimizing sample conditions for RPA/ssDNA complexes prior to study by SAXS and/or SANS.
MeSH term(s)	DNA Replication ; DNA, Single-Stranded/metabolism ; DNA-Binding Proteins/chemistry ; DNA-Binding Proteins/metabolism ; Molecular Biology/methods ; Neutron Diffraction/methods ; Replication Protein A/chemistry ; Replication Protein A/metabolism ; Scattering, Small Angle ; X-Ray Diffraction/methods
Chemical Substances	DNA, Single-Stranded ; DNA-Binding Proteins ; Replication Protein A
Language	English
Publishing date	2012-09-14
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-62703-032-8_6
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Article ; Online: Targeting SARS-CoV-2 Nsp3 macrodomain structure with insights from human poly(ADP-ribose) glycohydrolase (PARG) structures with inhibitors.

Brosey, Chris A / Houl, Jerry H / Katsonis, Panagiotis / Balapiti-Modarage, Lakshitha P F / Bommagani, Shobanbabu / Arvai, Andy / Moiani, Davide / Bacolla, Albino / Link, Todd / Warden, Leslie S / Lichtarge, Olivier / Jones, Darin E / Ahmed, Zamal / Tainer, John A

Progress in biophysics and molecular biology

2021 Volume 163, Page(s) 171–186

Abstract: Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, ... ...

Abstract	Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono (ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy. We therefore solved the structure of CoV-2 Mac1 from non-structural protein 3 (Nsp3) and applied structural and sequence-based genetic tracing, including newly determined A. pompejana MacroD2 and GDAP2 amino acid sequences, to compare and contrast CoV-2 Mac1 with the functionally related human DNA-damage signaling factor poly (ADP-ribose) glycohydrolase (PARG). Previously, identified targetable features of the PARG active site allowed us to develop a pharmacologically useful PARG inhibitor (PARGi). Here, we developed a focused chemical library and determined 6 novel PARGi X-ray crystal structures for comparative analysis. We applied this knowledge to discovery of CoV-2 Mac1 inhibitors by combining computation and structural analysis to identify PARGi fragments with potential to bind the distal-ribose and adenosyl pockets of the CoV-2 Mac1 active site. Scaffold development of these PARGi fragments has yielded two novel compounds, PARG-345 and PARG-329, that crystallize within the Mac1 active site, providing critical structure-activity data and a pathway for inhibitor optimization. The reported structural findings demonstrate ways to harness our PARGi synthesis and characterization pipeline to develop CoV-2 Mac1 inhibitors targeting the ADP-ribose active site. Together, these structural and computational analyses reveal a path for accelerating development of antiviral therapeutics from pre-existing drug optimization pipelines.
MeSH term(s)	Amino Acid Sequence ; Antiviral Agents/chemistry ; Antiviral Agents/pharmacology ; Catalytic Domain ; Coronavirus Papain-Like Proteases/metabolism ; Crystallography, X-Ray ; Drug Discovery ; Enzyme Inhibitors/chemistry ; Enzyme Inhibitors/pharmacology ; Glycoside Hydrolases/antagonists & inhibitors ; Humans ; Models, Molecular ; Protein Domains ; SARS-CoV-2/drug effects ; SARS-CoV-2/metabolism ; Small Molecule Libraries/chemistry ; Small Molecule Libraries/pharmacology ; Structure-Activity Relationship ; Xanthines/chemistry ; Xanthines/pharmacology ; COVID-19 Drug Treatment
Chemical Substances	Antiviral Agents ; Enzyme Inhibitors ; Small Molecule Libraries ; Xanthines ; methylxanthine (28109-92-4) ; Glycoside Hydrolases (EC 3.2.1.-) ; poly ADP-ribose glycohydrolase (EC 3.2.1.143) ; Coronavirus Papain-Like Proteases (EC 3.4.22.2) ; papain-like protease, SARS-CoV-2 (EC 3.4.22.2)
Language	English
Publishing date	2021-02-23
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	209302-9
ISSN	1873-1732 ; 0079-6107
ISSN (online)	1873-1732
ISSN	0079-6107
DOI	10.1016/j.pbiomolbio.2021.02.002
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Article ; Online: Preparation of the modular multi-domain protein RPA for study by NMR spectroscopy.

Brosey, Chris A / Chagot, Marie-Eve / Chazin, Walter J

Methods in molecular biology (Clifton, N.J.)

2011 Volume 831, Page(s) 181–195

Abstract: The integrity and propagation of the genome depend upon the fidelity of DNA processing events, such as replication, damage recognition, and repair. Requisite to the numerous biochemical tasks required for DNA processing is the generation and manipulation ...

Abstract	The integrity and propagation of the genome depend upon the fidelity of DNA processing events, such as replication, damage recognition, and repair. Requisite to the numerous biochemical tasks required for DNA processing is the generation and manipulation of single-stranded DNA (ssDNA). As the primary eukaryotic ssDNA-binding protein, Replication Protein A (RPA) protects ssDNA templates from stray nuclease cleavage and untimely reannealment. More importantly, RPA also serves as a platform for organizing access to ssDNA for readout of the genetic code, recognition of aberrations in DNA, and processing by enzymes. We have proposed that RPA's ability to adapt to such a broad spectrum of multiprotein machinery arises in part from its modular organization and interdomain flexibility. While requisite for function, RPA's modular flexibility has presented many challenges to providing a detailed characterization of the dynamic architecture of the full-length protein. To enable the study of RPA's interdomain dynamics and responses to ssDNA binding by biophysical methods including NMR spectroscopy, we have successfully produced recombinant full-length RPA in milligram quantities at natural abundance and enriched with NMR-active isotopes.
MeSH term(s)	Cell Culture Techniques/methods ; Cell Transformation, Viral ; Chromatography, Gel ; Culture Media/chemistry ; DNA, Single-Stranded/metabolism ; Isotope Labeling/methods ; Nuclear Magnetic Resonance, Biomolecular/methods ; Protein Conformation ; Protein Structure, Tertiary ; Replication Protein A/biosynthesis ; Replication Protein A/chemistry ; Replication Protein A/isolation & purification ; Replication Protein A/metabolism
Chemical Substances	Culture Media ; DNA, Single-Stranded ; Replication Protein A
Language	English
Publishing date	2011-12-13
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-61779-480-3_11
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