A suite of 19 F based relaxation dispersion experiments to assess biomolecular motions
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ARTICLE
A suite of 19F based relaxation dispersion experiments to assess biomolecular motions Jan H. Overbeck1 · Werner Kremer1 · Remco Sprangers1 Received: 23 June 2020 / Accepted: 18 September 2020 © The Author(s) 2020
Abstract Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of 19F-based CPMG, on-resonance R1ρ and off-resonance R1ρ RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the 19F RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with 19F RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of 19F NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions. Graphic abstract
Keywords Fluorine · Large complexes · Protein folding · Relaxation dispersion · Structural dynamics.
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10858-020-00348-4) contains supplementary material, which is available to authorized users. * Remco Sprangers [email protected] 1
Department of Biophysics I, Regensburg Center for Biochemistry, University of Regensburg, 93053 Regensburg, Germany
Bio-molecules are inherently dynamic and populate a number of structurally different states. NMR spectroscopy is a unique tool to experimentally investigate these bio-molecular motions with atomic resolution. When the exchange rates between the different states are on the millisecond timescale Carr–Purcell–Meiboom–Gill (CPMG) and rotating-frame relaxation experiments at different effective magnetic fields can be exploited to record relaxation dispersion (RD) profiles. These profiles directly depend on thermodynamic
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(populations) and kinetic (rates) parameters of the exchange process and structural information (chemical shifts) of sparsely populated (invisible), short-lived excited states can be obtained. For proteins under 20 kDa, RD experiments can be recorded on protonated 15N-labeled samples, whereas deuteration and transverse relaxation optimized spectroscopy (TROSY; Pervushin et al. 1997) are required for larger systems. In very large complexes (> 80 kDa), methyl group labeling in a fully deuterated background combined with methyl TROSY based approaches (Tugarinov et al. 2003; Schütz and Sprangers 2019; Abramov et al. 2020) can be used
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