Understanding Protein Function Through an Ensemble Description: Characterization of Functional States by ¹⁹F NMR

Protein function is a consequence of a complex and dynamic equilibrium between allosterically coupled functional states. However, it is often difficult to distinguish the representative members of an ensemble by spectroscopic means. ¹⁹F NMR is particularly useful in this regard owing to the sensitivity of its chemical shift to subtle differences in environment. Here, we address aspects of ¹⁹F NMR relevant to the study of ensembles. In particular, we discuss current trends toward: (1) ¹⁹F-reporters that can be biosynthetically incorporated into proteins, (2) Approaches to chemical tagging of proteins by ¹⁹F reporters, (3) Improving delineation of states by ¹⁹F NMR, (4) Distinguishing states by (¹⁹F NMR-based) topology measurements that focus on solvent exposure and hydrophobicity, (5) Relaxation experiments and simple approaches to delineating states in fast and slow exchange, (6) Extending resolution of states by ¹⁹F NMR, and (7) Validating ¹⁹F NMR spectroscopy by computational methods. Many of these advances are demonstrated through recent ¹⁹F NMR studies of a homodimeric enzyme, fluoroacetate dehalogenase.