Measuring Diffusion Constants of Invisible Protein Conformers by Triple-Quantum 1 H CPMG Relaxation Dispersion.

Proteins are not locked in a single structure but often interconvert with other conformers that are critical for function. When such conformers are sparsely populated and transiently formed they become invisible to routine biophysical methods, however they can be studied in detail by NMR spin-relaxation experiments. Few experiments are available in the NMR toolkit, however, for characterizing the hydrodynamic properties of invisible states. Herein we describe a CPMG-based experiment for measuring translational diffusion constants of invisible states using a pulsed-field gradient approach that exploits methyl 1 H triple-quantum coherences. An example, involving diffusion of a sparsely populated and hence invisible unfolded protein ensemble is presented, without the need for the addition of denaturants that tend to destroy weak interactions that can be involved in stabilizing residual structure in the unfolded state.