Using hydroxyl radical footprinting to explore the free energy landscape of protein folding.
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 Φ-value analysis.
School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Antonio N
AN
Calabrese
School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
James R
JR
Ault
School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address: s.e.radford@leeds.ac.uk.
Sheena E
SE
Radford
School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address: a.e.ashcroft@leeds.ac.uk.
Alison E
AE
Ashcroft
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D017665:Hydroxyl Radical / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D017510:Protein Folding; D023302:Protein Footprinting / Q000379:methods*; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001426:Bacterial Proteins; D017665:Hydroxyl Radical
Electrospray ionisation-mass spectrometry; Fast photochemical oxidation of proteins; Hydroxyl radical footprinting; Liquid chromatography–mass spectrometry; Protein folding; Protein structure