Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution.

The physical basis of protein structure, dynamics and function has been intensely studied for several decades. Indeed since the new millennium there has been a tremendous expansion in the number of unique topological folds that have been characterized at high resolution by crystallographic and nuclear magnetic resonance (NMR) based methods. In the midst of this rush towards a grand scale structural genomics effort, a quieter effort dedicated to the experimental characterization of protein conformational heterogeneity has also emerged. The influence of atomic scale structure on molecular recognition and catalysis by proteins is often the focus of attention while the role of dynamics is largely unknown and frequently ignored. Nevertheless, it has long been recognized that proteins are indeed dynamic systems. Early insights into the time scale and character of protein internal motion largely employed local optical probes, unresolved hydrogen exchange, and one dimensional NMR techniques that, though limited, revealed a startling complexity and richness in the internal motion of proteins.1–5 These initial views contributed significantly to the development of current treatments of protein dynamics and thermodynamics.6 The connection to biological function rather than just biological form is more recent. Internal protein dynamics can potentially affect protein function through a variety of mechanisms, some of which are tautological or obvious in nature while others are subtle and remain to be fully explored and appreciated. There are now several examples of protein-protein and protein-ligand interactions that illustrate that dynamics may be fundamentally linked to function in several ways. Nuclear magnetic resonance (NMR) spectroscopy is very much at the center of current efforts to illuminate the nature of protein dynamics and their role in biological function. Here we will focus on the use of solution NMR methods to provide fast subnanosecond dynamics of protein side chains. The interested reader is referred to separate reviews in this issue by Goehlert and Stone,7 Tolman8 and Palmer and Massi9 discussing dynamics of the polypeptide backbone. The emerging success of NMR spectroscopy in the arena of protein dynamics rests on four general areas of development over the past two decades. First and perhaps foremost, triple resonance NMR spectroscopy now provides an efficient and robust set of tools for the comprehensive resonance assignment of proteins of significant size.10,11 These methods, in turn, derive much of their power from companion isotopic enrichment strategies,12,13 which have been subsequently refined to allow for isotopic labeling patterns that are optimized for NMR relaxation studies (vide infra). Two-dimensional sampling of relaxation has allowed for comprehensive studies to be efficiently undertaken, albeit with great instrumental cost.14–16 A variety of technical issues such as the effects of macromolecular tumbling and the influence of competing relaxation mechanisms have also been largely resolved (vide infra). These advances have positioned solution NMR spectroscopy to efficiently and comprehensively characterize the fast internal dynamics of proteins of significant size. This review seeks to provide a compact but reasonably complete description of the theoretical and technical foundation for solution NMR relaxation methods that are currently being brought to bear on fast sub-nanosecond protein side chain dynamics and to present a summary of current findings and their possible significance. A survey of basic observations about side chain dynamics derived from NMR-relaxation studies is presented along with several analyses meant to dispel commonly held but apparently inaccurate correlations between dynamics, structure and function. How dynamics can enter into fundamental thermodynamic and kinetic aspects of protein function is also reviewed and illustrated with intriguing results from several systems that point to a promising future for this area of inquiry.