| While hydrophobic amino acid residues are most often found in the interior of proteins, a number of proteins have been shown to have solvent exposed hydrophobic residues. These surface exposed hydrophobic sites in proteins are thought to be involved in interactions with small molecules, membrane lipids and other proteins, pertaining to their functions.; Polarity sensitive molecular probes have been used to characterize hydrophobic protein surfaces. The stoichiometry and binding constants of the fluorophore 1-anilinonaphthalene-8-sulfonate (ANSA) and chromophore bromophenol blue (BPB) to the model protein, BSA, are determined by fluorimetric and spectrophotometric titration, respectively. BPB absorption difference spectroscopy is demonstrated to be an alternative approach to fluorescence for identifying surface hydrophobic sites on proteins.; The establishment of a subproteomic library of expressed proteins whose native structures are typified by the presence of surface hydrophobic sites is proposed. It is demonstrated that proteins with hydrophobic surfaces can be detected selectively by staining with ANSA and BPB on an electrophoretic gel run under nondenaturing conditions to help elucidate new catalytic, transport and regulatory functionalities in complex proteomic screenings.; The binding of ANSA and BPB to hydrophobic sites on proteins has been applied to the study of drug binding to serum proteins. A rapid titration technique is proposed for the determination of a drug binding constant by displacement of a polarity sensitive fluorophore or chromophore that competes for the same hydrophobic site on a protein surface as the drug under investigation.; Homology modeling of the BSA binding site for ANSA and BPB is carried out based on a crystal structure of a close sequence neighbor, human serum albumin (HSA). Further, to help elucidate the binding mode of the polarity sensitive dye, BPB, to BSA and L-lactate dehydrogenase (LDH), flexible docking is attempted, using the Lamarckian genetic search algorithm. The preferred binding sites for the dye are found to be hydrophobic, and Gibbs free energy estimates are in agreement with the experimental equilibrium constants. |
