The Conformations of the DrkN SH3 Domain Studied by Single Molecule Fluorescence Spectroscopy

Abstract

SH3 domains are highly involved in signal transduction and cellular localization. The N-terminal SH3 domain of Drosophila adaptor protein Drk is found to be marginally stable, exchanging between folded and unfolded states under non-denaturating conditions. The high unfolded state population makes DrkN SH3 a useful model system to study the physical polymeric properties of disordered protein states and to advance the understanding of the mechanism of protein folding. Single-molecule techniques have the unique capability to resolve populations of protein conformations and also the rates of exchange dynamics among them. Here, single-molecule Föster resonance energy transfer (smFRET) is performed in order to study the conformational distribution and dynamics of the DrkN SH3 domain, using fluorophores attached to two cysteines mutated at the N- and the C-termini of the 61amino acid chain. Freely-diffusing proteins in diluted solutions give rise to fluorescence bursts which can be quantitatively characterized. Multiparameter fluorescence analysis reveals two populations with different end-to-end distances, attributed to the folded and unfolded states coexisting under normal conditions. Conformational populations and internal chain dynamics are measured in both physiological and non-physiological conditions in order to understand the role of solvent-protein interactions for the structural stability. Fluorescence correlation spectroscopy (FCS) and FRET-FCS are applied to investigate local chain dynamics and the inter-conversion kinetics between the ordered and disordered conformations of DrkN SH3 in different solvent conditions.