Isotope Labeling Methods for Relaxation Measurements

Lundström, Patrik; Ahlner, Alexandra; Blissing, Annica Theresia

doi:10.1007/978-94-007-4954-2_4

Cited by 3 publications

(3 citation statements)

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“…Protein dynamics on the millisecond time scale in aromatic side chains can be studied by 13 C (Weininger et al 2012) and 1 H (Raum et al 2018) CPMG relaxation dispersion experiments. The key requirement for 13 C relaxation dispersion experiments is site-selective 13 C labeling of aromatic side chains (Lundström et al 2012; Schörghuber et al 2018; Weininger 2019), which eliminates 1 J 13 C– 13 C couplings. To date, there are several well established labeling strategies, that achieve this goal (Kasinath et al 2013; Lichtenecker 2014; Lichtenecker et al 2013; Lundström et al 2007; Milbradt et al 2015; Schörghuber et al 2015, 2017a, b; Teilum et al 2006; Weininger 2017a, b).…”

Section: Introductionmentioning

confidence: 99%

Site-selective 1H/2H labeling enables artifact-free 1H CPMG relaxation dispersion experiments in aromatic side chains

et al. 2019

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Aromatic side chains are often key residues in enzyme active sites and protein binding sites, making them attractive probes of protein dynamics on the millisecond timescale. Such dynamic processes can be studied by aromatic 13C or 1H CPMG relaxation dispersion experiments. Aromatic 1H CPMG relaxation dispersion experiments in phenylalanine, tyrosine and the six-ring moiety of tryptophan, however, are affected by 3J 1H–1H couplings which are causing anomalous relaxation dispersion profiles. Here we show that this problem can be addressed by site-selective 1H/2H labeling of the aromatic side chains and that artifact-free relaxation dispersion profiles can be acquired. The method has been further validated by measuring folding–unfolding kinetics of the small protein GB1. The determined rate constants and populations agree well with previous results from 13C CPMG relaxation dispersion experiments. Furthermore, the CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained directly from the spectra. In summary, site-selective 1H/2H labeling enables artifact-free aromatic 1H CPMG relaxation dispersion experiments in phenylalanine and the six-ring moiety of tryptophan, thereby extending the available methods for studying millisecond dynamics in aromatic protein side chains.

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Section: Introductionmentioning

confidence: 99%

Site-selective 1H/2H labeling enables artifact-free 1H CPMG relaxation dispersion experiments in aromatic side chains

et al. 2019

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“…CPMG-based RD experiments have also been devised for 1 H and 13 C nuclei in the protein backbone, in protein side chains, along with relaxation-optimized experiments for probing high-molecular weight proteins [ 10 ]. In many of these experiments, scalar couplings are either suppressed experimentally or eliminated by use of specifically isotope-labeled protein samples [ 18 ]. With the exception of deuteration, which modulates the strengths of van der Waal's interactions and can, thus, have an effect on structural dynamics, isotope labeling is generally considered noninvasive.…”

Section: Relaxation Dispersion Experimentsmentioning

confidence: 99%

NMR Methods to Study Dynamic Allostery

2016

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Nuclear magnetic resonance (NMR) spectroscopy provides a unique toolbox of experimental probes for studying dynamic processes on a wide range of timescales, ranging from picoseconds to milliseconds and beyond. Along with NMR hardware developments, recent methodological advancements have enabled the characterization of allosteric proteins at unprecedented detail, revealing intriguing aspects of allosteric mechanisms and increasing the proportion of the conformational ensemble that can be observed by experiment. Here, we present an overview of NMR spectroscopic methods for characterizing equilibrium fluctuations in free and bound states of allosteric proteins that have been most influential in the field. By combining NMR experimental approaches with molecular simulations, atomistic-level descriptions of the mechanisms by which allosteric phenomena take place are now within reach.

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“…A key requirement for studies of protein dynamics, that are often directly linked to function (Mittermaier and Kay 2006 ), are isolated 1 H-X spin pairs that are not affected by coupling with their neighbours. While being the default for dynamic studies of backbone amides (Akke and Palmer 1996 ; Ishima and Torchia 2003 ; Jarymowycz and Stone 2006 ; Loria et al 1999 ), dynamics studies of amino acid side chains (Hansen and Kay 2011 ; Hansen et al 2012 ; Lundstrom et al 2009a ; Millet et al 2002 ; Muhandiram et al 1995 ; Mulder et al 2002 ; Paquin et al 2008 ; Weininger et al 2012a , c ) often requires site selective 13 C and/or 2 H labeling (Lundstrom et al 2012b ). Studies of side chain dynamics not only complement existing backbone studies, but widen the view on certain processes and enable unique additional information of structure (Korzhnev et al 2010 ; Neudecker et al 2012 ), ring-flips (Weininger et al 2014b ; Yang et al 2015 ), histidine tautomers (Weininger et al 2017 ) and proton occupancy and transfer reactions (Hansen and Kay 2014 ; Wallerstein et al 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Site-selective 13C labeling of histidine and tryptophan using ribose

Weininger

2017

J Biomol NMR

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Experimental studies on protein dynamics at atomic resolution by NMR-spectroscopy in solution require isolated 1H-X spin pairs. This is the default scenario in standard 1H-15N backbone experiments. Side chain dynamic experiments, which allow to study specific local processes like proton-transfer, or tautomerization, require isolated 1H-13C sites which must be produced by site-selective 13C labeling. In the most general way this is achieved by using site-selectively 13C-enriched glucose as the carbon source in bacterial expression systems. Here we systematically investigate the use of site-selectively 13C-enriched ribose as a suitable precursor for 13C labeled histidines and tryptophans. The 13C incorporation in nearly all sites of all 20 amino acids was quantified and compared to glucose based labeling. In general the ribose approach results in more selective labeling. 1-13C ribose exclusively labels His δ2 and Trp δ1 in aromatic side chains and helps to resolve possible overlap problems. The incorporation yield is however only 37% in total and 72% compared to yields of 2-13C glucose. A combined approach of 1-13C ribose and 2-13C glucose maximizes 13C incorporation to 75% in total and 150% compared to 2-13C glucose only. Further histidine positions β, α and CO become significantly labeled at around 50% in total by 3-, 4- or 5-13C ribose. Interestingly backbone CO of Gly, Ala, Cys, Ser, Val, Phe and Tyr are labeled at 40–50% in total with 3-13C ribose, compared to 5% and below for 1-13C and 2-13C glucose. Using ribose instead of glucose as a source for site-selective 13C labeling enables a very selective labeling of certain positions and thereby expanding the toolbox for customized isotope labeling of amino-acids.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-017-0130-9) contains supplementary material, which is available to authorized users.

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Isotope Labeling Methods for Relaxation Measurements

Cited by 3 publications

References 74 publications

Site-selective 1H/2H labeling enables artifact-free 1H CPMG relaxation dispersion experiments in aromatic side chains

Site-selective 1H/2H labeling enables artifact-free 1H CPMG relaxation dispersion experiments in aromatic side chains

NMR Methods to Study Dynamic Allostery

Site-selective 13C labeling of histidine and tryptophan using ribose

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