title: Structural organization and stability of a thermoresistant domain generated by in vivo hydrolysis of the alpha-crystallin B chain from calf lens.
abstract: A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry and Biophysics, University of Naples, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Russo
authors.forename: D
authors.initials: D
authors.lastname: Vincenti
authors.forename: R
authors.initials: R
authors.lastname: Ragone
authors.forename: P
authors.initials: P
authors.lastname: Stiuso
authors.forename: G
authors.initials: G
authors.lastname: Colonna
mesh_terms: D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007908:Lens, Crystalline / Q000737:chemistry; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D003459:Crystallins; D046911:Macromolecular Substances