Probing dynamics in single molecules

Abstract The ability to resolve accurate distance fluctuations on the single-molecule level concurrently with the timescale of the dynamics is vital to the understanding of many biological and biochemical reactions. Forster resonance energy transfer (FRET) in combination with single-molecule fluorescence microscopy has grown into one of the most popular tools to study the kinetics of such processes in real-time. Dynamics is the link that connects the function of a protein with its structure and function. The molecular mechanisms involved strongly depend on the conformation as well as inter- and intramolecular dynamics. In this chapter, we introduce the instrumentation, experimental method, software and data analysis required for studying surface-immobilized single molecules on the basis of FRET via total internal reflection fluorescence microscopy. After extraction of the single molecule FRET traces, the number of underlying conformational states and the transition rates between them can be determined using a hidden Markov model (HMM) analysis. We provide details of the HMM analysis method and demonstrate its usefulness in investigating conformational fluctuations of the TATA-box binding protein on DNA upon the addition of the transcription factor NC2.