CPMG relaxation dispersion NMR experiments measuring glycine 1Hα and 13Cα chemical shifts in the ‘invisible’ excited states of proteins
Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are extremely powerful for characterizing millisecond time-scale conformational exchange processes in biomolecules. A large number of such CPMG experiments have now emerged for measuring protein backbone chemical shifts of sparsely populated (>0.5%), excited state conformers that cannot be directly detected in NMR spectra and that are invisible to most other biophysical methods as well. A notable deficiency is, however, the absence of CPMG experiments for measurement of 1Hα and 13Cα chemical shifts of glycine residues in the excited state that reflects the fact that in this case the 1Hα, 13Cα spins form a three-spin system that is more complex than the AX 1Hα–13Cα spin systems in the other amino acids. Here pulse sequences for recording 1Hα and 13Cα CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which 1Hα, 13Cα chemical shifts can be obtained. The utility of these experiments is demonstrated by an application to a mutant of T4 lysozyme that undergoes a millisecond time-scale exchange process facilitating the binding of hydrophobic ligands to an internal cavity in the protein.
KeywordsCPMG Relaxation dispersion Excited protein states T4 lysozyme Millisecond dynamics
This work was supported by funds from the Canadian Institutes of Health Research (CIHR) in the form of a research grant to LEK and postdoctoral fellowships to DFH and PL (Protein Folding Training Grant). LEK holds a Canada Research Chair in Biochemistry.
- Bax A, Ikura M, Kay LE, Torchia DA, Tschudin R (1990) Comparison of different modes of 2-dimensional reverse-correlation NMR for the study of proteins. J Magn Reson 86:304–318Google Scholar
- Burum DP, Ernst RR (1980) Net polarization transfer via a J-ordered state for signal enhancement of low-sensitivity nuclei. J Magn Reson 39:163–168Google Scholar
- Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141Google Scholar
- Goddard TD, Kneller DG. SPARKY 3. University of California, San FranciscoGoogle Scholar
- Grey MJ, Tang Y, Alexov E, McKnight CJ, Raleigh DP, Palmer AG III (2006) Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain. J Mol Biol 355:1078–1094CrossRefGoogle Scholar
- Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins. J Magn Reson 85:393–399Google Scholar
- Mulder FA, Skrynnikov NR, Hon B, Dahlquist FW, Kay LE (2001b) Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123:967–975CrossRefGoogle Scholar
- Palmer AG, Cavanagh J, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected 2-dimensional heteronuclear correlation Nmr-spectroscopy. J Magn Reson 93:151–170Google Scholar
- Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broad-band decoupling-Waltz-16. J Magn Reson 52:335–338Google Scholar
- States DJ, Haberkorn RA, Ruben DJ (1982) A two-dimensional nuclear overhauser experiment with pure absorption phase in 4 quadrants. J Magn Reson 48:286–292Google Scholar