Reversed-phase liquid chromatography as a tool in the determination of the hydrophilicity/hydrophobicity of amino acid side-chains at a ligand-receptor interface in the presence of different aqueous environments. II. Effect of varying peptide ligand hydrophobicity.
The present study represents a continuation of our development of a chromatographic model for studying the hydrophobic interactions which characterize the way a ligand binds to its receptor. We have designed 18-residue amphipathic alpha-helical peptides (representing the hydrophobic binding domain of a ligand), where the non-polar face interacts with the non-polar face of a reversed-phase stationary phase (representing a receptor protein with a hydrophobic binding pocket). Two series of amphipathic alpha-helical peptides were subjected to reversed-phase liquid chromatography at pH 2.0, where the "native" Ala-face peptide contains seven Ala residues in its non-polar face and the "native" Leu-face series contains seven Leu residues in its non-polar face. Mutants of the two series were then prepared by replacing one residue in the centre of the non-polar face in both series of peptides, resulting in amino acid side-chains being exposed to a moderately non-polar environment (Ala series) or a very hydrophobic environment (Leu series) surrounding the substitution site. With this model, we have demonstrated that an increase in non-polarity of the ligand enhances hydrophilicity (decreases hydrophobicity) of all amino acids at the ligand-receptor interface, this effect being dependent on the intrinsic hydrophilicity/hydrophobicity of the side-chain. The addition of salt to the aqueous environment surrounding the binding site of the ligand and receptor was also shown to affect the hydrophilicity/hydrophobicity of amino acids in the binding interface. For the Ala-face mutants, the majority of the non-polar side-chains and the three positively charged residues (Arg, His, Lys) showed significant enhancement of hydrophobicity in the presence of salt; in contrast, in the much more hydrophobic environment of the Leu-face mutants, there was a trend of lesser hydrophobicity enhancement and/or significantly more hydrophilicity enhancement in the presence of salt. Our results should have major implications for the understanding of the hydrophilicity/ hydrophobicity of side-chains in varying hydrophobic and aqueous environments.
Mant
Colin T
CT
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver 80262, USA.
Hodges
Robert S
RS
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008024:Ligands; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D008024:Ligands; D010455:Peptides; D011956:Receptors, Cell Surface