Structure and dynamics of protein excited states with millisecond lifetimes

Abstract

An in-depth understanding of biological processes often requires detailed atomic resolution structures of the molecules involved. However in solution where most of these processes occur the conformation of biomolecules like RNA, DNA and proteins is not static but fluctuates. Routinely used structural techniques like X-ray crystallography, NMR spectroscopy and cryo-electron microscopy have almost always been used to determine the structure of the dominant conformation or obtain an average structure of the biomolecule in solution with very little detailed information regarding the dynamics of these molecules in solution. Over the last few years, NMR based methods have been developed to study the dynamics of these biomolecules in solution in a site-specific manner with the aim of generating structures of the different conformations that these molecules can adopt in solution. One powerful technique is the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment, which can be used to detect and characterize protein excited states that are populated for as less as 0.5% of the time with ∼0.5–10 millisecond lifetimes. Due to recent advances in NMR pulse sequences and labeling methodology, it is now possible to determine the structures of these transiently populated excited states with millisecond lifetimes by obtaining accurate chemical shifts, residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) of these excited states. In these excited states the dynamics of some methyl containing residues can also be studied.