Probing invisible, low-populated States of protein molecules by relaxation dispersion NMR spectroscopy: an application to protein folding.

Biological function depends on molecular dynamics that lead to excursions from highly populated ground states to much less populated excited states. The low populations and the transient formation of such excited states render them invisible to the conventional methods of structural biology. Thus, while detailed pictures of ground-state structures of biomolecules have emerged over the years, largely through X-ray diffraction and solution nuclear magnetic resonance (NMR) spectroscopy studies, much less structural data has been accumulated on the conformational properties of the invisible excited states that are necessary to fully explain function. NMR spectroscopy is a powerful tool for studying conformational dynamics because it is sensitive to dynamics over a wide range of time scales, extending from picoseconds to seconds and because information is, in principle, available at nearly every position in the molecule. Here an NMR method for quantifying millisecond time scale dynamics that involve transitions between different molecular conformations is described. The basic experimental approach, termed relaxation dispersion NMR spectroscopy, is outlined to provide the reader with an intuitive feel for the technology. A variety of different experiments that probe conformational exchange at different sites in proteins are described, including a brief summary of data-fitting procedures to extract both the kinetic and thermodynamic properties of the exchange process and the structural features of the invisible excited states along the exchange pathway. It is shown that the methodology facilitates detection of intermediates and other excited states that are populated at low levels, 0.5% or higher, that cannot be observed directly in spectra, so long as they exchange with the observable ground state of the protein on the millisecond time scale. The power of the methodology is illustrated by a detailed application to the study of protein folding of the small modular SH3 domain. The kinetics and thermodynamics that describe the folding of this domain have been characterized through the effects of temperature, pressure, side-chain deuteration, and mutation, and the structural features of a low-populated folding intermediate have been assessed. Despite the fact that many previous studies have shown that SH3 domains fold via a two-state mechanism, the NMR methods presented unequivocally establish the presence of an on-pathway folding intermediate. The unique capabilities of NMR relaxation dispersion follow from the fact that large numbers of residues can be probed individually in a single experiment. By contrast, many other forms of spectroscopy monitor properties that are averaged over all residues in the molecule or that make use of only one or two reporters. The NMR methodology is not limited to protein folding, and applications to enzymatic catalysis, binding, and molecular recognition are beginning to emerge.