Abstract A 2D 13C Chemical Exchange Saturation Transfer (CEST) experiment is presented for studying slowly exchanging protein systems using methyl groups as probes. The utility of the method is first established through studies of protein L, a small protein, for which chemical exchange on the millisecond time-scale is not observed. Subsequently the approach is applied to a folding exchange reaction of a G48M mutant Fyn SH3 domain, for which only cross-peaks derived from the folded (â??groundâ??) state are present in spectra. Fits of 15N and methyl 13C CEST profiles of the Fyn SH3 domain establish that the exchange reaction involves an interchange between folded and unfolded conformers, although elevated methyl 13C transverse relaxation rates for some of the residues of the unfolded (â??invisible, excitedâ??) state indicate that it likely exchanges with a third conformation as well. In addition to the kinetics of the exchange reaction, methyl carbon chemical shifts of the excited state are also obtained from analysis of the 13C CEST data.
Content Type Journal Article
Category Article
Pages 1-8
DOI 10.1007/s10858-012-9640-7
Authors
Guillaume Bouvignies, Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON M5S 1A8, Canada
Lewis E. Kay, Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON M5S 1A8, Canada
Measurement of the signs of methyl 13C chemical shift differences between interconverting ground and excited protein states by R1Ď?: an application to αB-crystallin
Measurement of the signs of methyl 13C chemical shift differences between interconverting ground and excited protein states by R1Ď?: an application to αB-crystallin
Abstract Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG RD) NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond time-scale exchange processes involving the interconversion between a visible ground state and one or more minor, sparsely populated invisible â??excitedâ?? conformational states. Recently it has also become possible to determine atomic resolution...
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Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supramolecular Protein Systems: Applications to the Proteasome and to the ClpP Protease
Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supramolecular Protein Systems: Applications to the Proteasome and to the ClpP Protease
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http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja202259a/aop/images/medium/ja-2011-02259a_0002.gif
Journal of the American Chemical Society
DOI: 10.1021/ja202259a
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Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supra-Molecular Protein Systems: Applications to the Proteasome and to the ClpP Protease.
Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supra-Molecular Protein Systems: Applications to the Proteasome and to the ClpP Protease.
Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supra-Molecular Protein Systems: Applications to the Proteasome and to the ClpP Protease.
J Am Chem Soc. 2011 May 11;
Authors: Religa TL, Ruschak AM, Rosenzweig R, Kay LE
Methyl groups are powerful reporters of structure, motion and function in NMR studies of supra-molecular protein assemblies. Their...
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05-12-2011 03:40 PM
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Chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance phenomenon that can be used to probe the solvent-accessibility of tryptophan, tyrosine, and histidine residues in proteins by means of laser-induced photochemical reactions, resulting in significant enhancement of NMR signals. CIDNP offers good sensitivity as a surface probe of...
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Direct spin trapping studies of protein radical adducts are limited as a consequence of the long rotational correlation times and consequent broadening of the ESR resonances. It can be difficult to determine both the nature and number of...
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Ph.D. student in solid-state NMR: Studying protein folding and assembly at atomic sca
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Application of the random coil index to studying protein flexibility
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Abstract:
Protein flexibility lies at the heart of many protein–ligand binding events and enzymatic activities. However, the experimental measurement of protein motions is often difficult, tedious and error-prone. As a result, there is a considerable interest in developing simpler and faster ways of quantifying protein flexibility. Recently, we described a method, called Random Coil Index (RCI), which appears to be able to...
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Simultaneous detection of amide and methyl correlations using a time shared NMR experiment: application to binding epitope mapping
Peter Würtz, Olli Aitio, Maarit Hellman and Perttu Permi
Journal of Biomolecular NMR; 2007; 39(2) pp 97 - 105
Abstract:
Simultaneous recording of different NMR parameters is an efficient way to reduce the overall experimental time and speed up structural studies of biological macromolecules. This can especially be beneficial in the case of fast NMR-based drug screening applications or for collecting NOE restraints, where prohibitively long data collection...
A 2D 13C-CEST experiment for studying slowly exchanging protein systems using methyl probes: an application to protein folding
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