Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy

Abstract

Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)-ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of 15 N, 1 HN, 13 C ␣ , and 13 CO chemical shifts, along with 1 HN-15 N, 1 H ␣ -13 C ␣ , and 1 HN-13 CO residual dipolar couplings and 13 CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with 1 HN-15 N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins.Carr-Purcell-Meiboom-Gill (CPMG) ͉ dynamics ͉ excited state structure ͉ residual dipolar couplings ͉ chemical exchange M uch of structural biology focuses on the generation of static structures of biomolecules, with less consideration of how such structures change in time. This, in part, reflects the sample requirements that are imposed by the different structural methods. In the case of studies using x-ray diffraction, for example, molecular dynamics are minimized due to the crystalline nature of the sample itself, which traps the molecule in a single conformation, and by the liquid nitrogen temperatures that are used in most experiments. In the case of solution NMR spectroscopy, sample conditions are sought that give rise to the ''highest quality spectra'' that often derive from the most stable form of the protein, and conformational heterogeneity that reflects excursions between different states is thus minimized. Certainly, initial focus on a single static representation is a wise choice, as much can be learned from these high-resolution pictures. However, function is often predicated on excursions between different conformers (1, 2), and it is therefore of interest to obtain detailed structural information about the many different states of a biomolecule that populate its energy landscape.Of the different conformers that one wishes to study, it is clear that those belonging to the low energy ground state are the most easily probed, because they are populated to the greatest extent. Hig...

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