Experimental Evidence for Millisecond-Timescale Structural Evolution Following the Microsecond-Timescale Folding of a Small Protein

Prior work has shown that small proteins can fold (i.e., convert from unstructured to structured states) within 10 mu s. Here we use time-resolved solid state nuclear magnetic resonance (ssNMR) methods to show that full folding of the 35-residue villin headpiece subdomain (HP35) requires a slow annealing process that has not been previously detected. C-13 ssNMR spectra of frozen HP35 solutions, acquired with a variable time tau(e) at 30 degrees C after rapid cooling from 95 degrees C and before rapid freezing, show changes on the 3-10 ms timescale, attributable to slow rearrangements of protein sidechains during tau(e).