Unfolding/Folding Of A Multi-Domain Protein Untangled By Single-Molecule Fret

Deciphering how the amino acid code translates into 3D structures is the key to understand how proteins really work. In this context, the two-domain protein phosphoglycerate kinase (PGK) has proven to be an excellent model for multi-domain proteins. It is known, that both domains of PGK interact during folding. The N-terminal domain only gains its native structure in presence of the C-terminal domain. The C-domain also folds individually but the process is facilitated by the N-domain. In addition, intermediate states are involved. [1,2] A detailed picture of these intermediates and to what extent they are populated is still missing. It is therefore challenging to unravel the mechanisms of tertiary structure formation, especially since subpopulations are hard to identify in ensemble methods. To avoid averaging over all conformations, single-molecule methods are a perfect tool to distinguish and quantify such subpopulations. We established a set of PGK cysteine variants for site-specific labeling with fluorescent dyes for single molecule fluorescence resonance energy transfer (FRET). We verified that secondary and tertiary structures were not affected by cysteine mutations applying circular dichroism (CD) spectroscopy and tryptophan fluorescence. In addition all PGK cysteine mutants were catalytically active. The native states of the double labelled PGK variants were thoroughly characterized by fluorescence correlation spectroscopy (FCS) and single molecule FRET. Our system is designed to follow motions in between and within the individual domains displayed by distance changes of fluorophores during unfolding/folding transitions under denaturing conditions.[1] S. Osvath, J. J. Sabelko, M. Gruebele, J Mol Biol 2003, 333(1):187.[2] J.-H. Han, S. Batey, A. A. Nickson, S. A. Teichmann, J. Clarke, Nat Rev Mol Cell Bio 2007, 8:319.