| The results obtained on the unfolding and stability of the leucine specific (LS-) and leucine-isoleucine-valine (LIV) binding proteins (BP) and mutants (LSW18F, LSW278F, LSW320F, LSW336F, and LIVY18W) are presented. These two wild-type periplasmic binding proteins from Escherichia coli have a high degree of sequence and structural homology and have overlapping specificities for branched chain amino acids. Two spectroscopic techniques were used to investigate protein unfolding and stability of LS- and LIV-BP: (1) fluorescence and (2) second derivative ultraviolet spectroscopy. The two state and three state models used to describe the urea induced equilibrium unfolding of these proteins in the absence and presence of ligand are discussed. ΔG's were in the range of 34.7 to 47.0 kJ/mol. In addition, we investigated the effect of incorporation of fluorinated amino acid analogues, 5F-tryptophan, 3F-phenylalanine, 4F-phenylalanine, and difluoromethionine, on stability. These fluorinated amino acids can serve as 19F NMR probes of protein structure and function. The urea induced unfolding curves of both unlabeled wild-type binding proteins in the absence of ligand were sigmoidal and single phase. These data can be described by and fitted to either a two-state or three-state model in which folded proteins were in equilibrium with unfolded proteins. In the presence of ligand, the midpoints of the transition curves for both the LS-BP and LIV-BP increased with increasing ligand concentrations and the curves were biphasic, indicating that the bound protein complex undergoes a three state unfolding and the protein-ligand complex has greater conformational stability than the empty binding protein. The stability of LIV-BP with ligands (leucine, isoleucine, valine, serine and threonine) showed the complex with isoleucine to be most stable, being more stable than the empty protein by 41.5 kJ/mol. The stability of the mutant proteins W278F and W320F (with the replacement of a tryptophan with a phenylalanine in the proteins hydrophobic core) are less stable than the wild-type. Finally, the replacement of aromatic amino acids in LS-BP with fluorinated ones does not significantly affect the protein's native stability, and in the case of 5-fluorotryptophan the stability is, on average, increased. |
