title: Reconstructing NMR spectra of "invisible" excited protein states using HSQC and HMQC experiments.
abstract: Carr-Purcell-Meiboom-Gill (CPMG) relaxation measurements employing trains of 180 degrees pulses with variable pulse spacing provide valuable information about systems undergoing millisecond-time-scale chemical exchange. Fits of the CPMG relaxation dispersion profiles yield rates of interconversion, relative populations, and absolute values of chemical shift differences between the exchanging states, |Deltaomega|. It is shown that the sign of Deltaomega that is lacking from CPMG dispersion experiments can be obtained from a comparison of chemical shifts in the indirect dimensions in either a pair of HSQC (heteronuclear single quantum coherence) spectra recorded at different magnetic fields or HSQC and HMQC (heteronuclear multiple quantum coherence) spectra obtained at a single field. The methodology is illustrated with an application to a cavity mutant of T4 lysozyme in which a leucine at position 99 has been replaced by an alanine, giving rise to exchange between ground state and excited state conformations with a rate on the order of 1450 s(-1) at 25 degrees C.
authors.affiliation: Protein Engineering Network Center of Excellence, Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8. nikolai@purdue.edu
authors.forename: Nikolai R
authors.initials: NR
authors.lastname: Skrynnikov
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Dahlquist
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution; D017122:Bacteriophage T4 / Q000201:enzymology; D007930:Leucine / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*
chemical_list: D009113:Muramidase; D007930:Leucine; D000409:Alanine