title: Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion.
abstract: Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use (15)N, , and (13)CO NMR R(2) relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R(2) dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75-4.95 and determined to be an on-pathway intermediate (I1) in a linear three-state unfolding scheme (N&lrarr2;I1&lrarr2;MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N → I1 (36 s(-1)) and MG → I1 (26 s(-1)), respectively. Differences in chemical shift between N and I1 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R(2) dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N → MG transition. The experiments also identify regions of energetic frustration that "crack" during unfolding and impede the refolding process.
authors.affiliation: Department of Molecular Biology and Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla CA 92037, USA.
authors.forename: Derrick W
authors.initials: DW
authors.lastname: Meinhold
authors.forename: Peter E
authors.initials: PE
authors.lastname: Wright
mesh_terms: D000577:Amides / Q000737:chemistry; D001059:Apoproteins / Q000737:chemistry; D002138:Calibration; D019295:Computational Biology / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008968:Molecular Conformation; D009211:Myoglobin / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013696:Temperature
chemical_list: D000577:Amides; D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin