Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion

Weininger, Ulrich; Respondek, Michal; Akke, Mikael

doi:10.1007/s10858-012-9656-z

Cited by 41 publications

(76 citation statements)

References 58 publications

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“…13 C relaxation dispersion experiments both for CPMG (Weininger et al 2012b) and R 1ρ (Weininger et al 2014a) were validated for glucose labeled samples previously. These experiments can be directly applied to samples resulting from erythrose labeling, since the relaxation behaviour is identical.…”

Section: Resultsmentioning

confidence: 99%

Site-selective 13C labeling of proteins using erythrose

Weininger

2017

J Biomol NMR

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NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13C and/or 1H, which is achieved in the most general way by using site-selectively 13C-enriched glucose (1- and 2-13C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively 13C-enriched erythrose (1-, 2-, 3- and 4-13C) as a suitable precursor for 13C labeled aromatic side chains. We quantify 13C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the 13C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated 13C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective 13C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-017-0096-7) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Site-selective 13C labeling of proteins using erythrose

Weininger

2017

J Biomol NMR

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“…Accordingly, methods have been developed for 1 H N [87-89], 1 H α [90-92], 13 CO [93-95], 13 C α [92,94,96], and 13 C β [97] spins. Related methods have been developed for aromatic groups, which are relatively rare in proteins but provide additional probes of the hydrophobic core dynamics [98] and side chain 1 H spins [18]. …”

Section: 13c and 1h Relaxation Dispersionmentioning

confidence: 99%

Chemical exchange in biomacromolecules: Past, present, and future

Palmer

2014

Journal of Magnetic Resonance

239

220

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The perspective reviews quantitative investigations of chemical exchange phenomena in proteins and other biological macromolecules using NMR spectroscopy, particularly relaxation dispersion methods. The emphasis is on techniques and applications that quantify the populations, interconversion kinetics, and structural features of sparsely populated conformational states in equilibrium with a highly populated ground state. Applications to folding, mol ecular recognition, catalysis, and allostery by proteins and nucleic acids are highlighted.

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“…It is now possible in some cases to measure scalar and residual dipolar couplings that, in turn, provide additional structural insights (15). Further advances have led to the measurement of side-chain 13 C and 1 H chemical shifts in invisible states (16)(17)(18) that can be sensitive to hydrophobic contacts in these rare conformers. Experiments for quantifying pico-to nanosecond time-scale side-chain dynamics have also emerged (19), showing in some cases large differences in motion between ground and excited states that directly relate to function (20).…”

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confidence: 99%

Measuring hydrogen exchange rates in invisible protein excited states

Long

Bouvignies

Kay

2014

Proc. Natl. Acad. Sci. U.S.A.

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Hydrogen exchange rates have become a valuable probe for studying the relationship between dynamics and structure and for dissecting the mechanism by which proteins fold to their native conformation. Typically measured rates correspond to averages over all protein states from which hydrogen exchange can occur. Here we describe a new NMR experiment based on chemical exchange saturation transfer that provides an avenue for obtaining uncontaminated, per-residue amide hydrogen exchange rates for interconverting native and invisible states so long as they can be separated on the basis of distinct 15 N chemical shifts. The approach is applied to the folding reaction of the Fyn SH3 domain that exchanges between a highly populated, NMR-visible native state and a conformationally excited, NMR-invisible state, corresponding to the unfolded ensemble. Excellent agreement between experimentally derived hydrogen exchange rates of the excited state at a pair of pHs is obtained, taking into account the expected dependence of exchange on pH. Extracted rates for the unfolded ensemble have been used to test hydrogen exchange predictions based on the primary protein sequence that are used in many analyses of solvent exchange rates, with a Pearson correlation coefficient of 0.84 obtained.amide exchange with solvent | conformationally excited protein states | protein folding T he energy landscape of a protein is a multidimensional surface composed of many local minima in addition to the global minimum that is the native conformation (1, 2). An understanding of the relation between protein structure, dynamics, and function is predicated, therefore, on an analysis of the various conformational states that populate the minima on the landscape. This requires detailed structural and dynamics studies of each of the conformers and quantification of their relative energies as well as the kinetics of exchange between them. Biophysical techniques such as X-ray diffraction and NMR spectroscopy are available for obtaining detailed structural information on the molecules populating the lowest-energy regions of the landscape, providing insight into the structure-function paradigm for a great number of proteins. However, it is becoming increasingly well understood that studies of the ground states of proteins are not sufficient. Additional states that can be sparsely populated and transiently formed, referred to as excited states in what follows, are often important for processes that include molecular recognition, ligand binding, enzyme catalysis, and folding (3-9). Detailed studies of such excited states are, however, challenging because they are not "visible" to standard biophysical methods and as a consequence atomic resolution information is lacking.Recent developments in solution NMR spectroscopy are changing this paradigm by providing an avenue for quantifying excited states at a level of detail that has typically only been possible in studies of highly populated native protein conformers (10). Backbone 1 H, 13 C, and 15 N chemical shifts fo...

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Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion

Cited by 41 publications

References 58 publications

Site-selective 13C labeling of proteins using erythrose

Site-selective 13C labeling of proteins using erythrose

Chemical exchange in biomacromolecules: Past, present, and future

Measuring hydrogen exchange rates in invisible protein excited states

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