Nanospray HX-MS configuration for structural interrogation of large protein systems

Sheff, Joey; Hepburn, Morgan; Yu, Yaping; Lees‐Miller, Susan P.; Schriemer, David C.

doi:10.1039/c6an02707e

Cited by 22 publications

(24 citation statements)

References 31 publications

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“…Other approaches, such as HDX-MS, may be particularly useful in probing solvent exposed regions or regions of flexibility. Indeed, in preliminary studies, the region containing the ABCDE/T2609 phosphorylation cluster was highly accessible to deuterium, consistent with the presence of an exposed region of the protein (Sheff, Hepburn, Yu, Lees-Miller, & Schriemer, 2017). …”

Section: Dna Double-strand Break Responsesmentioning

confidence: 58%

What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses

Brosey

Ahmed

Lees‐Miller

et al. 2017

Methods in Enzymology

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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.

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Section: Dna Double-strand Break Responsesmentioning

confidence: 58%

What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses

Brosey

Ahmed

Lees‐Miller

et al. 2017

Methods in Enzymology

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“…Kinetics data from Sheff J.G. et al were mined to determine the protection factors for peptides in the central cavity of DNA-PKcs (Sheff et al, 2017). To distinguish structured from unstructured peptides, we calculated an average exchange rate constant k HX from timecourse data (0.5, 1, 5, 15 and 60 min) and referenced these values against the calculations of rate constants for the corresponding unstructured peptides according to the equation S SSEThread = S SS + S LL + S XL + S Tem + S Com + S SeMet , where P f represents the averaged protection factor for a given peptide i , k ch the averaged exchange rate for the unstructured form of the peptide, calculated according to (Bai et al, 1993), and k HX the measured average exchange rate constant for peptide i .…”

Section: Methodsmentioning

confidence: 99%

SSEThread: Integrative threading of the DNA-PKcs sequence based on data from chemical cross-linking and hydrogen deuterium exchange

Saltzberg

Hepburn

Pilla

et al. 2019

Progress in Biophysics and Molecular Biology

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X-ray crystallography and electron microscopy maps resolved to 3–8 angstroms are generally sufficient for tracing the path of the polypeptide chain in space, while often insufficient for unambiguously registering the sequence on the path (i.e., threading). Frequently, however, additional information is available from other biophysical experiments, physical principles, statistical analyses, and other prior models. Here, we formulate an integrative approach for sequence assignment to a partial backbone model as an optimization problem, which requires three main components: the representation of the system, the scoring function, and the optimization method. The method is implemented in the open source Integrative Modeling Platform (IMP) (https://integrativemodeling.org), allowing a number of different terms in the scoring function. We apply this method to localizing the sequence assignment within a 199-residue disordered region of three structured and sequence unassigned helices in the DNA-PKcs crystallographic structure, using chemical crosslinks, hydrogen deuterium exchange, and sequence connectivity. The resulting ensemble of threading models provides two major solutions, one of which suggests that the crucial ABCDE cluster of phosphorylation sites cannot undergo intra-molecular autophosphorylation without a conformational rearrangement. The ensemble of solutions embodies the most accurate and precise sequence threading given the available information.

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“…One explanation for this could be that the γ sub-unit exerts mechanical stress on the α3β3 sub-unit, and when under a PMF load, the C-terminal portion of the γ sub-unit over-twists, causing hydrogen bonds to destabilize ( Figure 9 b-d). Another study performed HDX-MS using a modified nanospray configuration to characterize DNA-PKcs, a 469 kDa protein, with 90% sequence coverage [ 60 ]. This configuration can be particularly useful for the study of proteins that have a higher complexity, and that cannot be produced in large quantities.…”

Section: Applications Of Hdx-msmentioning

confidence: 99%

HDX-MS: An Analytical Tool to Capture Protein Motion in Action

2020

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Virtually all protein functions in the cell, including pathogenic processes, require coordinated motion of atoms or domains, i.e., conformational dynamics. Understanding protein dynamics is therefore critical both for drug development and to learn about the underlying molecular causes of many diseases. Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) provides valuable information about protein dynamics, which is highly complementary to the static picture provided by conventional high-resolution structural tools (i.e., X-ray crystallography and structural NMR). The amount of protein required to carry out HDX-MS experiments is a fraction of the amount required by alternative biophysical techniques, which are also usually lower resolution. Use of HDX-MS is growing quickly both in industry and academia, and it has been successfully used in numerous drug and vaccine development efforts, with important roles in understanding allosteric effects and mapping binding sites.

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Nanospray HX-MS configuration for structural interrogation of large protein systems

Cited by 22 publications

References 31 publications

What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses

What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses

SSEThread: Integrative threading of the DNA-PKcs sequence based on data from chemical cross-linking and hydrogen deuterium exchange

HDX-MS: An Analytical Tool to Capture Protein Motion in Action

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