Using hydroxyl radical footprinting to explore the free energy landscape of protein folding.

Methods : a Companion to Methods in Enzymology
Antonio N CalabreseAlison E Ashcroft

Abstract

Characterisation of the conformational states adopted during protein folding, including globally unfolded/disordered structures and partially folded intermediate species, is vital to gain fundamental insights into how a protein folds. In this work we employ fast photochemical oxidation of proteins (FPOP) to map the structural changes that occur in the folding of the four-helical bacterial immunity protein, Im7. Oxidative footprinting coupled with mass spectrometry (MS) is used to probe changes in the solvent accessibility of amino acid side-chains concurrent with the folding process, by quantifying the degree of oxidation experienced by the wild-type protein relative to a kinetically trapped, three-helical folding intermediate and an unfolded variant that lacks secondary structure. Analysis of the unfolded variant by FPOP-MS shows oxidative modifications consistent with the species adopting a solution conformation with a high degree of solvent accessibility. The folding intermediate, by contrast, experiences increased levels of oxidation relative to the wild-type, native protein only in regions destabilised by the amino acid substitutions introduced. The results demonstrate the utility of FPOP-MS to characterise protein variant...Continue Reading

References

Nov 19, 2008·Mass Spectrometry Reviews·Vanessa Leah Mendoza, Richard W Vachet
Mar 12, 2011·Protein Engineering, Design & Selection : PEDS·Stuart KnowlingSheena E Radford
Dec 19, 2012·Biochimica Et Biophysica Acta·A KonijnenbergF Sobott

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Citations

Feb 14, 2016·Analytical and Bioanalytical Chemistry·Aimee RinasLisa M Jones
Nov 11, 2015·Methods : a Companion to Methods in Enzymology·Carol E Parker, Christoph H Borchers
Apr 14, 2016·Journal of the American Society for Mass Spectrometry·Siavash Vahidi, Lars Konermann
May 12, 2016·Biophysical Journal·Rachel M Abaskharon, Feng Gai
Jun 28, 2016·Journal of the American Society for Mass Spectrometry·Thomas G WatkinsonAlison E Ashcroft
Mar 10, 2017·Molecular & Cellular Proteomics : MCP·Liwen Wang, Mark R Chance
Mar 15, 2018·Annual Review of Biophysics·Janna Kiselar, Mark R Chance
Jun 20, 2020·Protein Science : a Publication of the Protein Society·Bob SchiffrinAntonio N Calabrese
Sep 26, 2020·Journal of Magnetic Resonance Imaging : JMRI·Haiqi YeKejia Cai
May 16, 2017·Journal of Magnetic Resonance Imaging : JMRI·Rong-Wen TainKejia Cai
Feb 16, 2021·Journal of the American Society for Mass Spectrometry·Owen CornwellAlison E Ashcroft
Nov 7, 2019·Analytical Chemistry·Owen CornwellAlison E Ashcroft
May 28, 2019·Journal of Proteome Research·Jeddidiah Bellamy-Carter, Neil J Oldham
Feb 11, 2020·Analytical Chemistry·Niloofar Abolhasani Khaje, Joshua S Sharp

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Methods Mentioned

BETA
protein folding
circular dichroism
NMR

Software Mentioned

MassLynx
FPOP
Driftscope
PEAKS Studio

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