Single Molecule Studies Of Ubiquitin Unfolding

Our group has recently reported the use of a specially designed single molecule nanomixer1 to study the unfolding kinetics of a yellow fluorescent protein (YFP)2 based on the measurements of single-pair fluorescence resonance energy transfer (spFRET), between the intrinsic chromophore and a covalently attached dye, and single-molecule fluorescence two-colour coincidence detection (TCCD). In this work we extend the application of the technology to the small and fast folding ubiquitin doubly labelled for spFRET and TCCD measurements. We firstly explored a variety of labelling strategies and fluorophores for optimal double labelling of ubiquitin, a key issue in application of single molecule fluorescence methods to protein folding/unfolding. We also added a second micropipette to the nanomixer for double-jump experiments to monitor single-molecule refolding. Using this dual labelled sample we have then studied the unfolding and refolding of ubiquitin at the single molecule level.References:[1] S. S. White, S. Balasubramanian, D. Klenerman, L. Ying Angew. Chem. Int. Ed. 2006, 45, 7540-7543.[2] A. Orte, T. D. Craggs, S. S. White, S. E. Jackson, D. Klenerman. J. Am. Chem. Soc., 130 (25), 7898-7907, 2008.