Exchange saturation transfer and associated NMR techniques for studies of protein interactions involving high-molecular-weight systems.

A brief overview of theoretical and experimental aspects of the Dark state Exchange Saturation Transfer (DEST) and lifetime line broadening ([Formula: see text]) NMR methodologies is presented from a physico-chemical perspective. We describe how the field-dependence of [Formula: see text] can be used for determining the exchange regime on the transverse spin relaxation time-scale. Some limitations of DEST/[Formula: see text] methodology in applications to molecular systems with intermediate molecular weights are discussed, and the means of overcoming these limitations via the use of closely related exchange NMR techniques is presented. Finally, several applications of DEST/[Formula: see text] methodology are described from a methodological viewpoint, with an emphasis on providing examples of how kinetic and relaxation parameters of exchange can be reliably extracted from the experimental data in each particular case.

Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR.

The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr-Purcell-Meinboom-Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the "invisible" GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from (15)N, (1)HN, and (13)Cmethyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.

NMR methods for exploring 'dark' states in ligand binding and protein-protein interactions.

A survey, primarily based on work in the authors' laboratory during the last 10 years, is provided of recent developments in NMR studies of exchange processes involving protein-ligand and protein-protein interactions. We start with a brief overview of the theoretical background of Dark state Exchange Saturation Transfer (DEST) and lifetime line-broadening (ΔR2) NMR methodology. Some limitations of the DEST/ΔR2 methodology in applications to molecular systems with intermediate molecular weights are discussed, along with the means of overcoming these limitations with the help of closely related exchange NMR techniques, such as the measurements of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, exchange-induced chemical shifts or rapidly-relaxing components of relaxation decays. Some theoretical underpinnings of the quantitative description of global dynamics of proteins on the surface of very high molecular weight particles (nanoparticles) are discussed. Subsequently, several applications of DEST/ΔR2 methodology are described from a methodological perspective with an emphasis on providing examples of how kinetic and relaxation parameters for exchanging systems can be reliably extracted from NMR data for each particular model of exchange. Among exchanging systems that are not associated with high molecular weight species, we describe several exchange NMR-based studies that focus on kinetic modelling of transient pre-nucleation oligomerization of huntingtin peptides that precedes aggregation and fibril formation.