Ever since its initial development, solution NMR spectroscopy has been used as a tool to study conformational exchange. Although many systems are amenable to relaxation dispersion approaches, cases involving highly skewed populations in slow chemical exchange have, in general, remained recalcitrant to study. Here an experiment to detect and characterize "invisible" excited protein states in slow exchange with a visible ground-state conformation (excited-state lifetimes ranging from ∼5 to 50 ms) is presented. This method, which is an adaptation of the chemical exchange saturation transfer (CEST) magnetic resonance imaging experiment, involves irradiating various regions of the spectrum with a weak B(1) field while monitoring the effect on the visible major-state peaks. The variation in major-state peak intensities as a function of frequency offset and B(1) field strength is quantified to obtain the minor-state population, its lifetime, and excited-state chemical shifts and line widths. The methodology was validated with (15)N CEST experiments recorded on an SH3 domain-ligand exchanging system and subsequently used to study the folding transition of the A39G FF domain, where the invisible unfolded state has a lifetime of ∼20 ms. Far more accurate exchange parameters and chemical shifts were obtained than via analysis of Carr-Purcell-Meiboom-Gill relaxation dispersion data.

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers)1–3. However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules4 and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta7 model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

Despite their importance for biological activity, slower molecular motions beyond the nanosecond range remain poorly understood. We have assembled an unprecedented set of experimental NMR data, comprising up to 27 residual dipolar couplings per amino acid, to define the nature and amplitude of backbone motion in protein G using the Gaussian axial fluctuation model in three dimensions. Slower motions occur in the loops, and in the ␤-sheet, and are absent in other regions of the molecule, including the ␣-helix. In the ␤-sheet an alternating pattern of dynamics along the peptide sequence is found to form a long-range network of slow motion in the form of a standing wave extending across the ␤-sheet, resulting in maximal conformational sampling at the interaction site. The alternating nodes along the sequence match the alternation of strongly hydrophobic side chains buried in the protein core. Confirmation of the motion is provided through extensive crossvalidation and by independent hydrogen-bond scalar coupling analysis that shows this motion to be correlated. These observations strongly suggest that dynamical information can be transmitted across hydrogen bonds and have important implications for understanding collective motions and long-range information transfer in proteins.protein dynamics ͉ slow motions ͉ correlated M olecular dynamics, manifest in backbone and side-chain mobilities, play a crucial role in protein stability and function (1-4). The accurate characterization and understanding of protein motions thus adds an additional dimension to the structural information derived from genomics projects (5, 6). Although local backbone fluctuations on the picosecond to nanosecond time scale have been the subject of detailed characterization using NMR (7, 8) and molecular dynamics simulations (2), slower motions, in the submicrosecond to second range, remain poorly understood. Relaxation dispersion has been used to successfully identify sites of conformational exchange between states experiencing different chemical shifts in peptides (9) and proteins (10), but specific geometric motional models are often difficult to extract from these data. Slow time scales are, however, of particular interest because functionally important biological processes, including enzyme catalysis (11), signal transduction (12), ligand binding, and allosteric regulation (13), as well as collective motions involving groups of atoms or whole amino acids (14), are expected to occur in this time range. Residual dipolar couplings (RDCs) report on averages over longer time scales (up to the millisecond range) and therefore encode key information for understanding slower protein motions over a very broad time scale (15,16). Recent studies have exploited the orientational averaging properties of RDCs to characterize the amplitude and direction of motions of NH vectors (17)(18)(19) or to study local variations in position and dynamics of the amide proton (20,21). Despite this activity, key questions remain concerning the nature and amplitude of ...