Single-molecule fluorescence studies of enzyme kinetics and protein-nucleic acid interactions.

摘要

Single-molecule methods can reveal many features of biological processes that are obscured by traditional ensemble averaged measurements. Thus they are useful for studying the molecular mechanisms of complicated biochemical reactions. In this thesis I present the applications of single-molecule fluorescence resonance energy transfer (FRET) to several biological systems that involve multistep enzymatic reactions and dynamic protein-nucleic acid interactions.;Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Using the two-way junction hairpin ribozyme as a model system, we demonstrated a method combining single-molecule FRET with Mg2+ pulse-chase experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic "fingerprint"). This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme-substrate complex.;Many essential biological reactions involve protein-nucleic acid interactions. Single-molecule FRET allows these reactions to be directly observed in real time, which helps the understanding of the relationship between the structural dynamics of the nucleoprotein complexes and their functions. We studied a key enzyme in the life cycle of human immunodeficiency virus (HIV), reverse transcriptase (RT), which encounters various nucleic acid substrates and catalyzes a series of reactions to convert single-stranded viral RNA into double-stranded DNA for host-cell integration. Our single-molecule FRET assays revealed that RT is a highly dynamic enzyme that can spontaneously flip between two binding orientations and slide over long distances on nucleic acid duplexes. These large-scale orientational and translational dynamics facilitate multiple phases of reverse transcription. This type of dynamic flexibility may be a general design principle for multifunctional enzymes like HIV RT, helping them to rapidly access different binding configurations required to accomplish different functions.