Measurement of signs of chemical shift differences between ground and excited protein
 

Abstract Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible â??excitedâ?? conformers. Analysis of CPMG data-sets also provides the magnitudes of the chemical shift difference(s) between exchanging states (|Î?Ï?|), that inform on the structural properties of the excited state(s). The sign of Î?Ï? is, however, not available from CPMG data. Here we present one-dimensional NMR experiments for measuring the signs of 1HN and 13Cα Î?Ï? values using weak off-resonance R 1Ï? relaxation measurements, extending the spin-lock approach beyond previous applications focusing on the signs of 15N and 1Hα shift differences. The accuracy of the method is established by using an exchanging system where the invisible, excited state can be converted to the visible, ground state by altering conditions so that the signs of Î?Ï? values obtained from the spin-lock approach can be validated with those measured directly. Further, the spin-lock experiments are compared with the established H(S/M)QC approach for measuring the signs of chemical shift differences. For the Abp1p and Fyn SH3 domains considered here it is found that while H(S/M)QC measurements provide signs for more residues than the spin-lock data, the two different methodologies are complementary, so that combining both approaches frequently produces signs for more residues than when the H(S/M)QC method is used alone.

• Content Type Journal Article
• DOI 10.1007/s10858-009-9394-z
• Authors
  • Renate Auer, University of Vienna Department of Structural and Computational Biology, Max F. Perutz Laboratories Campus-Vienna-Biocenter 5 1030 Vienna Austria
  • D. Flemming Hansen, University of Toronto Departments of Molecular Genetics, Biochemistry and Chemistry Toronto ON M5S 1A8 Canada
  • Philipp Neudecker, University of Toronto Departments of Molecular Genetics, Biochemistry and Chemistry Toronto ON M5S 1A8 Canada
  • Dmitry M. Korzhnev, University of Toronto Departments of Molecular Genetics, Biochemistry and Chemistry Toronto ON M5S 1A8 Canada
  • D. Ranjith Muhandiram, University of Toronto Departments of Molecular Genetics, Biochemistry and Chemistry Toronto ON M5S 1A8 Canada
  • Robert Konrat, University of Vienna Department of Structural and Computational Biology, Max F. Perutz Laboratories Campus-Vienna-Biocenter 5 1030 Vienna Austria
  • Lewis E. Kay, University of Toronto Departments of Molecular Genetics, Biochemistry and Chemistry Toronto ON M5S 1A8 Canada

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738
  • Journal Volume Volume 46
  • Journal Issue Volume 46, Number 3

Source: Journal of Biomolecular NMR