Measuring 1HN temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy
A method based on the Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for measuring the temperature coefficients of amide proton chemical shifts of low populated ‘invisible’ protein states that exchange with a ‘visible’ ground state on the millisecond time-scale. The utility of the approach is demonstrated with an application to an I58D mutant of the Pfl6 Cro protein that undergoes exchange between the native, folded state and a cold denatured, unfolded conformational ensemble that is populated at a level of 6% at 2.5°C. A wide distribution of amide temperature coefficients is measured for the unfolded state. The distribution is centered about –5.6 ppb/K, consistent with an absence of intra-molecular hydrogen bonds, on average. However, the large range of values (standard deviation of 2.1 ppb/K) strongly supports the notion that the unfolded state of the protein is not a true random coil polypeptide chain.
KeywordsCPMG relaxation dispersion Temperature coefficients Amide protons Cold denaturation
G.B. acknowledges the European Molecular Biology Organization and the Canadian Institutes of Health Research (CHIR) for post-doctoral fellowships. D.F.H was supported by a post-doctoral fellowship from the CIHR. This work was supported by grants from the CIHR and the Natural Sciences and Engineering Research Council of Canada (LEK) and the NIH (GM066806 to M.H.J.C.). L.E.K. holds a Canada Research Chair in Biochemistry.
- Anet FA, Basus VJ (1978) Limiting equations for exchaning broadening in 2-site NMR systmes with very unequal populations. J Magn Reson 32:339–343Google Scholar
- Dyson HJ, Rance M, Houghten RA, Lerner RA, Wright PE (1988) Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn. J Mol Biol 201:161–200Google Scholar
- Wishart DS, Case DA (2001) Use of chemical shifts in macromolecular structure determination. Methods Enzymol 338:3–34Google Scholar