Reconstructing NMR Spectra of “Invisible” Excited Protein States Using HSQC and HMQC Experiments

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Abstract
Carr−Purcell−Meiboom−Gill (CPMG) relaxation measurements employing trains of 180° 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, |Δω|. It is shown that the sign of Δω 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methodology is illustrated with an application to a cavity mutant of T4 lysozyme in which a leucine at position 99 has been replaced by an alanine, giving rise to exchange between ground state and excited state conformations with a rate on the order of 1450 s-1 at 25 °C.