Hydrophobic core mutations in CI2 globally perturb fast side-chain dynamics similarly without regard to position.
Protein dynamics is currently an area of intense research because of its importance as complementary information to the huge quantity of available data relating protein structure and function. Because it is usually the influence of dynamics on function that is studied, the physical determinants of the distribution of flexibility in proteins have not been explored as thoroughly. In the present NMR study, an expanded suite of five (2)H relaxation experiments was used to characterize the picosecond-to-nanosecond side-chain dynamics of chymotrypsin inhibitor 2 (CI2) and five hydrophobic core mutants, some of which are members of the folding nucleus. Because CI2 is a homologue of the serine protease inhibitor eglin c, which has already been extensively characterized in terms of its dynamics, it was possible to compare not only side-chain dynamics but also the responses of these dynamics to analogous mutations. Remarkably, each of the five core mutations in CI2 led to similar and reproducible increases in side-chain flexibility throughout the entire structure. Although the expanded suite of (2)H relaxation experiments does not affect model selection for the vast majority of residues, it did enable the detection of increasing levels of nanosecond-scale motions in CI2's reactive site binding loop as the L68 side chain was progressively shortened by mutation. Collectively, we observed that the CI2 mutants are more dynamically similar to each other than to the more rigid wild-type CI2, from which we propose that wild-type CI2 has been optimized to a specific level of rigidity which may aid in its function as a serine protease inhibitor. We also observed that the pattern of side-chain dynamics of CI2 is quantitatively similar to eglin c, but that this similarity is lost upon mutating both proteins at an equivalent position. Finally, (15)N relaxation was used to characterize the backbone dynamics of wild-type and mutant CI2. Interestingly, mutation at folding nucleus positions led to widespread increases in backbone flexibility, whereas non-folding-nucleus positions led to increases in flexibility in the C-terminal half of the protein only.
Whitley
Matthew J
MJ
Department of Biochemistry & Biophysics, School of Medicine, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Zhang
Jun
J
Lee
Andrew L
AL
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism
D010455:Peptides; D010940:Plant Proteins; D011506:Proteins; C093336:chymotrypsin inhibitor 2; C028656:eglin proteinase inhibitors