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, 1 H 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 W ithin the cell, unfolding and refolding of proteins occurs constantly, and spontaneous unfolding and misfolding processes play a central role in the formation of amyloid fibrils (1). In addition, fluctuations between native protein structure and partially or fully unfolded states have functional significance for binding, allosteric regulation, translocation across membranes, protein trafficking, secretion, and degradation (2). Despite the importance of spontaneous unfolding for protein function and cellular proteostasis, little is known about the transient, partially unfolded states that are formed. Detailed structural characterization of such states is difficult because of their inherently low populations and the conformational heterogeneity present when both native and partially unfolded states simultaneously exist in solution.Carr-Purcell-Meiboom-Gill (CPMG)-based R 2 relaxation dispersion experiments are unique in their ability to measure the kinetics of microsecond-millisecond exchange processes involving transient states with populations as sparse as 1%, and, in addition, allow determination of the associated NMR chemical shift changes (Δω) (3-5). In these experiments, the effective transverse relaxation rate R eff 2 is deconvoluted into the contribution from the exchange process, R ex , which varies with the pulsing frequency in the CPMG refocusing element, and the intrinsic relaxation rate R 0 2 . Here, we apply a series of amide and carbonyl R 2 dispersion experiments to investigate transient unfolding and refolding events within the native (N) state freeenergy landscape of apomyoglobin (apoMb).ApoMb is an ideal model system for studies of protein folding/ unfolding equilibria. The folded state is marginally stable at neutral pH, adopting a similar tertiary structure to the holoprotein except for disorder in the EF loop, F helix, FG loop, and N-terminal region of the G helix (residues 82-104), which appear to fluctuate between a folded native-like conformational substate and an ensemble of locally unfolded states (6, 7). ApoMb forms an equilibrium molten globule (MG) state at pH 4.1, amenable to direct investigation by NMR, which contains a significant number of native contacts and an overall compactness close to that of the N state (8-10). Kinetic refolding experiments show rapid formation of a burst-phase intermediate that resembles the equilibrium MG and contains native-like topology in the most structured regions (10-12). Folding to the N state from the MG is the overall ra...

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

“…Ligands apparently bind only to the WT′-like state, displaying conformational selection in ligand binding and, at the same time, indicating that the new states excited by the cavity have alternative packing arrangements that apparently fill or partially fill the cavity, a suggestion that is in line with recent studies in other proteins containing native or engineered cavities (22,(30)(31)(32)(33). Precedent for this behavior in T4L is found in the extensively studied cavity mutant L99A, where it has recently been shown that a poorly populated (≈3%) conformational substate in equilibrium with the WT is one in which the cavity is partially filled with a native phenylalanine side chain (31).…”

Protein dynamics and function from solution state NMR spectroscopy

“…Expansions to three-state models require data of outstanding quality (Sugase et al 2007) and complexity, such as results from experiments conducted at multiple static magnetic field strengths or temperatures. In very favorable cases a three state model can be used without additional constraints such as addition of ligands or change of temperature (Meinhold & Wright, 2011). There exist a few cases in which three-state models have successfully been applied to proteins undergoing exchange, such as the study by Grey et al (2003) on the multiple states induced by disulfide bond isomerization in the basic pancreatic trypsin inhibitor, and the calculation of structural ensembles of folding intermediates for two Fyn SH3 domain variants reported by Korzhnev et al (2004).…”

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

“…Moreover, heteronuclear NOE data reveal gradients of fast backbone fluctuations in these helices not observed in native apoMb (42). Relaxation dispersion NMR showed the MG to be in pH-dependent equilibrium with other partially folded forms, indicating that the MG state also will be present in an equilibrium mixture at neutral pH, although with a small population relative to the native structure (i.e., an excited state) (39). Furthermore, this equilibrium MG was found to be structurally similar to the second of two intermediates identified during kinetic refolding by ultrafast hydrogen/deuterium (H/D) exchange coupled with 2D NMR (43).…”

“…MGs typically are stabilized at low pH or in the presence of denaturants and are believed to represent intermediates in protein folding (38,39) and, in some cases, may mediate protein function (40). The MG of apomyoglobin (apoMb) stabilized at pH 4.1 is an ideal subject for exploring the structure and dynamics of such states because it can be populated under conditions suitable for study by high-resolution NMR (41).…”