Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry Follow Sub-millisecond Protein Folding at the Amino-Acid Level

摘要

We report a study of sub-millisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laserinduced temperature jump (T jump) from ~0 °C to ~ room temperature. Subsequent reactions via FPOP (fast photochemical oxidation of proteins) at various fractional millisecond points after the T jump leads to oxidative modification of solvent-accessible side chains whose “protection” changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix1 shows a decreasing trend of oxidative modification during the first 0.1-1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix1 formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the sub millisecond time range.