Optical spectroscopic approaches to study protein structure and dynamics
Our general approach is to use fluorescence spectroscopy to provide information on the solution structure, conformational dynamics, and ligand-protein interactions of bacterial toxins. We continue to employ both intrinsic and extrinsic probes for studying protein solution behaviour and ligand interaction. Trp is an excellent intrinsic fluorescence probe for studying the numerous protein events occurring in solution and/or in membranes because the Trp indole ring possesses a large molecular dipole moment and exhibits a pronounced change in its molecular dipole during the transition of the molecule to the singlet excited state.
This enables the study of dipole-dipole and specific interactions between the chromophore and the polar solvent molecules; in fact, large and highly solvent-dependent Stokes' shifts are observed for indoles. Hence, a single Trp residue located at a known site within the sequence of a protein provides a useful
fluorescent probe of protein-protein and protein-membrane events. We have used Trp fluorescence to study protein folding in a method used to determine site-specific folding events within proteins and to probe specific events occurring upon pH activation of toxins.
Our approach to use fluorescence to study proteins can be divided into the following areas:
- Steady state tryptophan fluorescence analysis of protein structure
- Stopped-flow analysis of ligand binding and enzyme-substrate interactions
- Time-resolved fluorescence of protein-protein and protein-ligand interactions
We have also extensively employed the method of site-directed fluorescence labelling of proteins with extrinsic fluorophores tethered to Cys residues within the primary sequence of the molecule. In order to facilitate this approach we first prepare a Cys-less mutant of our protein of interest by site-directed mutagenesis and then we determine the relative activity of the mutant protein compared with the wild-type. If the mutant activity is comparable to that of the wild-type then we can proceed to make a bank of single Cys mutants of the protein. Each mutant is chemically labelled at the Cys residue with a thiol-specific fluorophore such as monobromobimane (mBBr) or AEDANS. The stoichiometry of the labelling reaction is determined for each mutant-adduct and the fluorescence parameters of the tethered fluorophore are measured and used to correlate the structural and dynamic properties of the protein during its function. We have used this approach to study the membrane topology of the bacteriocin, colicin E1 and to study the nature of the protein-protein complex between toxins that possess mART enzyme activity and their protein substrates.
