title: The effect of phosphorylation on the structure of alpha-crystallin.
abstract: Homopolymers were constructed from the highly purified phosphorylated (A1 and B1) and non-phosphorylated (A2 and B2) polypeptides of alpha-crystallin. These were examined using electron microscopy, light scattering, fluorescence spectroscopy and sulphydryl probing to determine the effects of phosphorylation on the structure of alpha-crystallin. Each reconstituted aggregate consisted of uniform particles with circular cross-sections and diameters of 9.3-9.5 nm. Some of these were associated in chain-like structures. The molecular mass of the homopolymers varied from 360-507 kDa; for a single particle, it was estimated to be 216 +/- 10 kDa. Tryptophan residues in the alpha B2 homopolymers were more accessible to the solvent and to quenchers than those in alpha A2. Phosphorylation increased this accessibility in the alpha A homopolymer but decreased it in alpha B. This decrease could be attributed to phosphorylation of the serine adjacent to tryptophan 60 in the B chain. The kinetics of the reaction with DTNB indicated that the single cysteine in the A chain was buried in the alpha A2 homopolymer (k1' = 0.013 min-1) but more accessible in alpha A1 (k1' = 0.046 min-1). It was concluded that phosphorylation significantly alters the conformation of the alpha A subunits but probably has little effect on the B subunits. The alterations do not affect the ability of the subunits to associate into alpha-crystallin-like particles.
authors.affiliation: Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: R C
authors.initials: RC
authors.lastname: Augusteyn
authors.forename: J F
authors.initials: JF
authors.lastname: Koretz
authors.forename: P
authors.initials: P
authors.lastname: Schurtenberger
mesh_terms: D000818:Animals; D003459:Crystallins / Q000032:analysis* / Q000302:isolation & purification; D003545:Cysteine; D008854:Microscopy, Electron; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D011108:Polymers / Q000032:analysis; D011487:Protein Conformation; D012542:Scattering, Radiation; D012694:Serine; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
chemical_list: D003459:Crystallins; D010455:Peptides; D011108:Polymers; D012694:Serine; D014364:Tryptophan; D003545:Cysteine