文摘
An intrinsic steady-state fluorescent system for bovine adrenodoxin has been developed tostudy the protein structure in solution and the processes involved in protein unfolding. Since mature Adxcontains no natural Trp residue as internal probe, all of the aromatic amino acids, tyrosine at position 82and four phenylalanines at positions 11, 43, 59 and 64, were at each case replaced by tryptophan. Theresulting single tryptophan containing mutants kept their biological function compared with the wild type.Molecular modeling studies verify thermal unfolding experiments which point to a dramatically reducedstability caused by steric hindrance only for mutant F59W. Fluorescence spectra, Stern-Volmer quenchingconstants, and fluorescence energy transfer calculations indicated the analyzed positions to be situated insolution in the same immediate environment as in the crystal structure. Unfolding experiments with Gdn-HCl and time-resolved stopped-flow measurements provide evidence for differential stability and achronologically ordered unfolding mechanism of the different fluorescence probe positions in the protein.