Probing protein secondary structure and conformational heterogeneity.

Abstract

Time-resolved and steady-state fluorescence spectroscopy in conjunction with circular dichroism were employed to investigate secondary and tertiary structural features of proteins. Several single tryptophan (Trp) proteins with known crystal structures were examined both in solution and in the crystalline state. The three neurotoxins, $\alpha$-bungarotoxin, $\alpha$-cobratoxin and erabutoxin b, consist primarily of antiparallel $\beta$-sheet and contain a number of structurally important disulphide bridges. In each case, the Trp residue is situated approximately in the centre of a $\beta$-sheet. Complete random coil secondary structure for these proteins could be facilitated via the reduction and carboxymethylation or amidocarboxymethylation of the disulphide residues. Concurrent studies, by time-resolved fluorescence and circular dichroism, of these proteins at varied denaturant concentrations showed time-resolved fluorescence provides a highly sensitive probe of local secondary structure. Time-resolved fluorescence measurements for erabutoxin b single crystals exhibited multiexponential decay kinetics (single Trp residue). The relative proportion of each decay time component was found to be dependent upon the angular orientation of the protein crystal, with respect to the vertical polarization of the incident laser beam. These experimental data provide evidence for Trp side chain rotamers in the protein crystal. Model functions, which simulated the orientational dependence of the decay component relative proportions, were consistent with this rationalization. These results are the first direct experimental evidence obtained by fluorescence, to confirm the "rotamer" model for the interpretation of Trp fluorescence decay in solution. The measurement of crotonase, apoazurin and holoazurin crystals which display a range of exponential decay kinetics, provide further evidence for Trp rotamers in single protein crystals.