Visualizing transient dark states by NMR spectroscopy

Abstract

Myriad biological processes proceed through states that defy characterization by conventional atomic-resolution structural biological methods. The invisibility of these ‘dark’ states can arise from their transient nature, low equilibrium population, large molecular weight, and/or heterogeneity. Although they are invisible, these dark states underlie a range of processes, acting as encounter complexes between proteins and as intermediates in protein folding and aggregation. New methods have made these states accessible to high-resolution analysis by nuclear magnetic resonance (NMR) spectroscopy, as long as the dark state is in dynamic equilibrium with an NMR-visible species. These methods – paramagnetic NMR, relaxation dispersion, saturation transfer, lifetime line broadening, and hydrogen exchange – allow the exploration of otherwise invisible states in exchange with a visible species over a range of timescales, each taking advantage of some unique property of the dark state to amplify its effect on a particular NMR observable. In this review, we introduce these methods and explore two specific techniques – paramagnetic relaxation enhancement and dark state exchange saturation transfer – in greater detail.

Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR

“…2A), reflecting the interconversion between the F and I states, large differences in 15 N-R 2 between the free and GroELbound states of F and I, and differences in chemical shifts between the free and GroEL-bound I states. No dispersions were observed for SH3 WT (which is known to exist in a single folded state) in the presence of GroEL, indicating that the population of the GroELbound state (F-G) is too small and/or that the exchange between the F and F-G states likely occurs on a timescale faster (<50 μs) than can be accessed by CPMG experiments (16,19). The 15 N-DEST experiment involves the application of off-resonance radiofrequency (RF) radiation at a series of offsets from the resonances of the directly observable unligated F state to saturate 15 N nuclei in the large, spectroscopically invisible, "dark" GroEL-bound states (I-G and F-G) (18).…”

“…in solution as well as by the transverse relaxation rates of these species (SI Theory). Both lifetime line broadening and DEST rely on large differences in transverse relaxation rates between unligated and GroEL-bound species ( 15 N-R 2 ∼11 s −1 and ∼900 s −1 , respectively, at 900 MHz) (16), whereas relaxation dispersion is also dependent upon differences in chemical shifts between the interconverting states (19).…”

“…Moreover, hydrogen/deuterium exchange experiments on several protein substrates (9)(10)(11)(12) and fluorescence-based refolding experiments (13) suggest that the prototypical chaperonin GroEL may possess intrinsic unfoldase activity. Here we take advantage of a monomeric, nonaggregating, well-defined system-a triple mutant of the Fyn SH3 domain that exists in dynamic equilibrium between the major native state and a sparsely populated folding intermediate (14,15)-to directly demonstrate, using NMR relaxation-based methods (16), the ability of apo GroEL to accelerate the interconversion between these two states by almost three orders of magnitude. Simultaneous analysis of lifetime line-broadening (17), dark state exchange saturation transfer (DEST) (18), and Carr-Purcell-Meinboom-Gill (CPMG) relaxation dispersion (19) data permitted us to determine the catalytic rate constants and ascertain the location of the GroEL binding site on the folding intermediate under conditions where the population of GroEL-bound native and intermediate states is less than 1%.…”

Protein dynamics and function from solution state NMR spectroscopy

“…The exchange rate constant, k ex , must be much bigger than the difference between the relaxation rates of the two states; in practical terms, this means that k ex must be on the order of 1000 s −1 (Anthis & Clore, 2015). The magnitude of the experimentally determined PRE varies with the inverse sixth power of the distance, r, between the paramagnetic center and the studied nucleus:…”

Structural and Dynamic Insights of the Interaction between Tritrpticin and Micelles: An NMR Study

“…These states can be monitored by evaluating the HN indole region of the 1D-NMR spectrum, because it presents 12 peaks instead of three (i.e., one for each tryptophan residue). The presence of these peaks indicates a slow exchange regime between conformations in the chemicalshift timescale (43), rendering it possible to study the ensemble of the TRP3 peptide conformations by NMR. Fig.…”