Carr-Purcell-Meiboom-Gill Relaxation Dispersion Research Articles

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 (1)H(alpha) and (13)C(alpha) chemical shifts of glycine residues in the excited state that reflects the fact that in this case the (1)H(alpha), (13)C(alpha) spins form a three-spin system that is more complex than the AX (1)H(alpha)-(13)C(alpha) spin systems in the other amino acids. Here pulse sequences for recording (1)H(alpha) and (13)C(alpha) CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which (1)H(alpha), (13)C(alpha) 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.

The Folding Pathway of an FF domain: Characterization of an On-pathway Intermediate State Under Folding Conditions by 15N, 13C α and 13C-methyl Relaxation Dispersion and 1H/ 2H-exchange NMR Spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate ( I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by 15N, 13C α and methyl 13C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by 1H/ 2H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state ( F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by 15N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U↔ I↔ F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone 15N, 13C α and side-chain methyl 13C chemical shifts, with further information from protection factors for the backbone amide groups from 1H/ 2H-exchange. Notably, helices H1–H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl 13C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Φ-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Φ data can elucidate whole experimental folding pathways.

Reconstructing NMR spectra of "invisible" excited protein states using HSQC and HMQC experiments.

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.