Differential ordering of the protein backbone and side chains during protein folding revealed by site-specific recombinant infrared probes

摘要

The timescale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and side chains is particularly important for the formation of β-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/β structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone (¹³C=¹⁸O) using recombinant protein expression. T-jump, time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side chain dynamics with single residue resolution. We find that side chain ordering in a key region of the β-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely due to the transient formation of nonnative side chain interactions.