Journal Article

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

Meinhold, Derrick W., Wright, Peter E.

2011

Overview

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use ¹⁵N, ¹HN, and ¹³CO NMR R₂ 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 R₂ 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 onpathway intermediate (I1) in a linear three-state unfolding scheme (N ⇆ I1 ⇆ 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 R₂ 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.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Amides / Amides - chemistry / Apoproteins - chemistry / Biochemistry / Biological Sciences / Calibration / Cells / Chemical equilibrium / Computational Biology - methods / Correlations / Enzyme kinetics / High temperature / Hydrogen / Hydrogen-Ion Concentration / Kinetics / Magnetic Resonance Spectroscopy - methods / Models, Molecular / Molecular Conformation / Molecular structure / Myoglobin - chemistry / nitrogen / NMR / Nuclear magnetic resonance / nuclear magnetic resonance spectroscopy / Population parameters / Population structure / Protein Denaturation / Protein Folding / Protein refolding / Protein Structure, Secondary / Protein Structure, Tertiary / Protein Unfolding / stable isotopes / Temperature