We are studying the folding dynamics of a l85 repressor, the truncated N-terminal domain of l repressor, using single molecule measurements. The l85 repressor with double mutants (Gly46 ® Ala, Gly48 ® Ala) was reported to fold with a lifetime of less than 20ms (RE. Burton et al J.Mol.Biol 1996 263 311- 322) without intermediate states. The mutant protein, G46A/G48A, has been labeled with a fluorescent donor (Alexa488) and acceptor (Cy7) at its N and C termini. Using a Fluorescence Resonance Energy Transfer (FRET) measurement, we can investigate the dynamics of fast-folding proteins in two different environments, i) one in which a protein is confined but freely rotating in agarose gel, ii) the other in which a protein is fixated on glass surface through nitrilotriacetic acid (NTA) - chelated Nikel. In addition to the end-to-end distance distributions by the different urea concentrations, the instantaneous events during folding in a microsecond time scale and the fast-folding dynamics will be elucidated to explore the folding landscape and the memory effects of the G46A/G48A by applying novel single molecule spectroscopy analysis tool, Hidden Markov Model (HMM).
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