Measuring 1HN temperature coefficients in invisible protein states by relaxation dispersion NMR spectroscopy |
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| Authors: | Guillaume Bouvignies Pramodh Vallurupalli Matthew H J Cordes D Flemming Hansen Lewis E Kay |
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| Institution: | (1) Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON, M5S 1A8, Canada;(2) Department of Chemistry and Biochemistry, The University of Arizona, Tucson, AZ 85721-0088, USA;(3) Present address: Institute of Structural and Molecular Biology, University College London, London, WC1E 6BT, UK; |
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| Abstract: | 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. |
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