Fast Photochemical Oxidation
of Proteins and Mass
Spectrometry Follow Submillisecond Protein Folding at the Amino-Acid
Level
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Abstract
We report a study of submillisecond 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 laser-induced temperature jump (T jump)
from ∼0 °C to ∼room temperature. Subsequent reactions
via fast photochemical oxidation of proteins (FPOP) at various fractional
millisecond points after the T jump lead 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, helix<sub>1</sub> 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 helix<sub>1</sub> formed in a folding
intermediate but also show the efficacy of this new approach to following
protein folding on the submillisecond time range