A simple method to measure protein side-chain mobility using NMR chemical shifts.

Protein side-chain motions are involved in many important biological processes including enzymatic catalysis, allosteric regulation, and the mediation of protein-protein, protein-DNA, protein-RNA, and protein-cofactor interactions. NMR spectroscopy has long been used to provide insights into the motions of side-chain groups. Currently, the method of choice for studying side-chain dynamics by NMR is the measurement of methyl (2)H autorelaxation. Methyl (2)H autorelaxation exhibits simple relaxation mechanisms and can be straightforwardly converted to meaningful dynamic parameters. However, methyl groups can only be found in 6 of 19 side-chain bearing amino acids. Consequently, only a sparse assessment of protein side-chain dynamics is possible. Therefore, there is a significant interest in developing novel methods of studying side-chain motions that can be applied to all types of side-chains. Here, we show how side-chain chemical shifts can be used to determine the magnitude of fast side-chain motions in proteins. The chemical shift method is applicable to all side-chain bearing residues and does not require any additional measurements beyond standard NMR experiments for backbone and side-chain assignments.

[1]  A. Szabó,et al.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity , 1982 .

[2]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[3]  A. Szabó,et al.  Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes. , 1980, Biophysical journal.

[4]  A. Wand,et al.  Main chain and side chain dynamics of a heme protein: 15N and 2H NMR relaxation studies of R. capsulatus ferrocytochrome c2. , 2001, Biochemistry.

[5]  David S Wishart,et al.  A simple method to predict protein flexibility using secondary chemical shifts. , 2005, Journal of the American Chemical Society.

[6]  Main chain and side chain dynamics of oxidized flavodoxin from Cyanobacterium anabaena. , 2001, Biochemistry.

[7]  R. R. Ernst,et al.  A Protocol for the Interpretation of Side-Chain Dynamics Based on NMR Relaxation: Application to Phenylalanines in Antamanide , 1997 .

[8]  David S Wishart,et al.  NMR: prediction of protein flexibility , 2006, Nature Protocols.

[9]  K. Pearson.  Mathematical Contributions to the Theory of Evolution. III. Regression, Heredity, and Panmixia , 1896 .

[10]  G A Petsko,et al.  Fluctuations in protein structure from X-ray diffraction. , 1984, Annual review of biophysics and bioengineering.

[11]  Andrew E. Torda,et al.  The GROMOS biomolecular simulation program package , 1999 .

[12]  G. Lipari.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules , 1982 .

[13]  Jane Clarke,et al.  What contributions to protein side-chain dynamics are probed by NMR experiments? A molecular dynamics simulation analysis. , 2005, Journal of molecular biology.

[14]  David S. Wishart,et al.  Application of the random coil index to studying protein flexibility , 2008, Journal of biomolecular NMR.

[15]  D. Woessner.  Spin Relaxation Processes in a Two‐Proton System Undergoing Anisotropic Reorientation , 1962 .

[16]  L. Kay,et al.  Analysis of deuterium relaxation-derived methyl axis order parameters and correlation with local structure , 1999, Journal of biomolecular NMR.

[17]  A. Wand,et al.  Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution. , 2006, Chemical reviews.

[18]  K. Kinosita,et al.  A theory of fluorescence polarization decay in membranes. , 1977, Biophysical journal.

[19]  Peter M. Kasson,et al.  GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit , 2013, Bioinform..

[20]  L. Kay,et al.  Correlation between 2H NMR side-chain order parameters and sequence conservation in globular proteins. , 2003, Journal of the American Chemical Society.

[21]  C. Spearman.  The proof and measurement of association between two things. By C. Spearman, 1904. , 1987, The American journal of psychology.

[22]  K. Teilum,et al.  Functional aspects of protein flexibility , 2009, Cellular and Molecular Life Sciences.

[23]  K. Constantine,et al.  Backbone and side chain dynamics of uncomplexed human adipocyte and muscle fatty acid-binding proteins. , 1998, Biochemistry.