title: Studies on the location of aromatic amino acids in alpha-crystallin.
abstract: The locations of tryptophan residues in alpha-crystallin and homopolymers constructed from the alpha A- and alpha B-chains were examined by comparing their fluorescence emission properties and their accessibilities to quenchers. Two classes of tryptophan could be distinguished on the basis of differences in their spectral characteristics, fluorescence decay lifetimes, quenching with acrylamide and exposure by increasing concentrations of urea. Polarization measurements indicated that the tryptophan residues were associated with flexible segments of the polypeptide chains. The two classes could be assigned, one to Trp-9 (in both A- and B-chains) which is in an hydrophobic environment, and one to Trp-60 (B-chain) which appeared to be nearer the surface of the aggregate. No evidence was found for residues inaccessible to the quenchers. An apparent partition coefficient of 40 was obtained for the association of acrylamide with the protein. From temperature-dependence studies, it was concluded that there was a significant energy barrier to the penetration of acrylamide into the protein matrix (Ea = 5.8 kcal/mol) and that entry of the quencher was through channels produced by temporary disruption of the matrix (delta s = 1.5 eu). The phenolic side-chains of tyrosine residues in several different alpha-crystallins were found to ionize with pK values above pH 11, indicative of residues highly shielded from the solvent. Comparisons of polypeptide sequences, together with tyrosine fluorescence emission data and the pK values, permitted a tentative assignment of residue locations. All of the data are consistent with a possible micelle-like structure for alpha-crystallin but not with a layered structure.
authors.affiliation: National Vision Research Institute of Australia, Carlton, Victoria.
authors.forename: R C
authors.initials: RC
authors.lastname: Augusteyn
authors.forename: K P
authors.initials: KP
authors.lastname: Ghiggino
authors.forename: T
authors.initials: T
authors.lastname: Putilina
mesh_terms: D020106:Acrylamide; D000178:Acrylamides; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007454:Iodides; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D014364:Tryptophan / Q000032:analysis*; D014508:Urea
chemical_list: D000178:Acrylamides; D000596:Amino Acids; D003459:Crystallins; D007454:Iodides; D020106:Acrylamide; D014364:Tryptophan; D014508:Urea