Bridging the gap between protein structure and dynamics: Single-molecule studies of protein conformational dynamics

Conformational dynamics are a ubiquitous aspect of protein structure. In solution, proteins are constantly bombarded by solvent molecules causing spontaneous transitions which span a wide range of length and timescales. The modern view of protein structure is a rough energy landscape on which many low energy configurations interconvert, rather than a singular low energy folded state. This raises the possibility that proteins have evolved to utilize these fluctuations in order to function. Verification of this hypothesis is challenging due to the averaging in conventional ensemble based experiments. In this work, high-resolution single-molecule Forster resonance energy transfer (FRET) spectroscopy is used to detect protein conformational dynamics on the functionally relevant ms-min timescale. Single-molecule FRET is uniquely suited to the study of conformational dynamics since it can measure both the timescale and magnitude of conformational fluctuations. This application of FRET relies on newly developed advanced statistical data analysis tools which explicitly account for the photon-counting noise that is pervasive in single-molecule experiments. The newly developed methods are characterized on single-molecule data collected on short poly-proline peptides. The relationship between conformational dynamics and function is studied in two proteins: E. coli Adenylate Kinase and M. tuberculosis PtpB. Both proteins are found to be highly flexable with significantly populated, well resolved conformational states interconverting on the millisecond timescale. A mechanism for Adenylate Kinase is proposed in which conformational dynamics constitute the rate limiting step for the enzyme's mechanism. Single-molecule studies of PtpB reveal a previously unobserved open conformation for a novel lid domain. A mechanism is proposed by which PtpB's lid serves as a dynamic filter to protect the enzyme's active site from oxidative inactivation within host macrophages.