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
Highlights
Bio-molecules are inherently dynamic and populate a number of structurally different states
In very large complexes (> 80 kDa), methyl group labeling in a fully deuterated background combined with methyl transverse relaxation optimized spectroscopy (TROSY) based approaches (Tugarinov et al 2003; Schütz and Sprangers 2019; Abramov et al 2020) can be used to record 13C single quantum (SQ; Skrynnikov et al 2001; Lundström et al 2007; Rennella et al 2016) and 13C multiple quantum (MQ; Korzhnev et al 2004) as well as 1H SQ (Tugarinov and Kay 2007; Baldwin et al 2010; Otten et al 2010; Weininger et al 2012), double quantum (DQ; Gopalan et al 2018) and triple quantum (TQ; Gopalan et al 2018) relaxation dispersion (RD) profiles
We developed a suite of one-dimensional CPMG, on-resonance R1ρ and off-resonance R1ρ pulse sequences for 19F nuclei
Summary
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. 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 to record 13C single quantum (SQ; Skrynnikov et al 2001; Lundström et al 2007; Rennella et al 2016) and 13C multiple quantum (MQ; Korzhnev et al 2004) as well as 1H SQ (Tugarinov and Kay 2007; Baldwin et al 2010; Otten et al 2010; Weininger et al 2012), double quantum (DQ; Gopalan et al 2018) and triple quantum (TQ; Gopalan et al 2018) RD profiles
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