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

作者：Calabrese Antonio N; Ault James R; Radford Sheena E^*; Ashcroft Alison E

来源：Methods, 2015, 89: 38-44.

DOI：10.1016/j.ymeth.2015.02.018

• Hydroxyl radical footprinting
• Fast photochemical oxidation of proteins
• Protein structure
• Protein folding
• Electrospray ionisation-mass spectrometry
• Liquid chromatography-mass spectrometry

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 variants in different conformational states and to provide insights into protein folding mechanisms that are complementary to measurements such as hydrogen/deuterium exchange labelling and Phi-value analysis.

• 出版日期2015-11-1