Primary Structure of the Bovine β‐Crystallin Bp Chain

Abstract

The major polypeptide chain of bovine 0-crystallin, BBp, was fragmented by means of cyanogen bromide treatment and by enzymatic digestions. Manual and automated Edman degradation of the resulting peptides provided the complete amino acid sequence of the 0Bp chain. The N-terminal alanine residue was shown to be N-aacetylated by mass spectrometry. The chain has a length of 204 residues and a calculated molecular weight of 23210. There is a considerable degree of homology between the N-terminal and C-terminal halves of the chain, presumably reflecting a tandem duplication of a shorter ancestral gene. The sequence of 0Bp is sufficiently related to that of y-crystallin I1 to place these proteins in the same superfamily. No sequence relationship was found with the a-crystallin chains.The water-soluble eye lens proteins, the crystallins, have proven to be suitable tools for the study of protein biosynthesis, differentiation, aging and molecular evolution [I]. For these purposes a knowledge of the primary stuctures of the crystallins is required, and these have indeed already been established for the a-crystallin A and B chains of several species [2 -41 and for the bovine y-crystallin fraction I1 [5]. We now have dctermined for the calf the complete amino acid sequence of the major polypeptide from the rcmaining class of mammalian crystallins, the 0-crystallin Bp chain. A preliminary report of some of the conclusions of this study has been published [6]. MATERIALS A N D METHODSIsolation and Fragmentation of [IBp /%Crystallin was isolated from calf lenses, and the major polypeptide /IBp purified as previously described [7, 81. The purity of 0Bp was checked by sodium dodecylsulfate/ polyacrylamide electrophoresis and alkaline urea gel electrophoresis [8].S-fi-Aminoethylation [9], carboxymethylation [lo] and cleavage with cyanogen bromide [I 11 (in 70 formic acid and using equal weights of protein and CNBr) were carried out according to standard procedures. Citraconylation of the &-amino groups of lysine [12] was carried out on cyanogen bromide fragments of carboxymethylated PBp.Digestions with trypsin (Worthington TRTPCK), chymotrypsin (Calbiochem A grade) and elastase (Whatman) were carried out in 0.1 M ammonium bicarbonate, brought to pH 8.9 with 1 M ammonia, at 37 'C for 90 min. Substrate concentrations were 10 mg/ml for protein or CNBr fragments, and 1 pmol/ml for peptides. The enzyme:substrate ratio was 2: 100 (w/w), or 0.5 mg/pmol in the case of peptides. Digestion with pepsin (Sigma) was carried out in 0.01 M HCI at a peptide concentration of 1 pmol/ml, using 0.5 mg of enzyme/pmol, during 5 h at 37 'C. Digestion with Stap1zylococcu.s uureus protcase (Miles V 8) was performed at 37 C for 30 h in 0.5 M ammonium bicarbonate, pH 7.8, at a protein concentration of 7 mg/ml and an enzyme : substrate ratio of 2 : 100 (w/w). Digests were either lyophilized (in case of trypsin after lowering the pH to 6.5 with 0.5 M HCl), or directly applied onto a gel filtration column for peptide separation. Purification of PeptidesCNBr fr...

Heat‐induced changes in the conformation of α‐ and β‐crystalline: Unique thermal stability of α‐crystallin

“…y-Crystallins, on the other hand, form aggregates at temperatures higher than their melting temperature [27]. pB2 shows 29% sequence homology with bovine y-11 [28]. X-ray studies [29-311 also suggest that a structural similarity exists between pS2 and y-11 crystallin.…”

1H‐NMR spectroscopy of bovine lens β‐crystallin

“…Based on their sequence similarity with the y-crystallins, it was proposed that each P-crystallin subunit consisted of four 'Greek key' motifs folded into two domains with the Nterminal globular domain of all P subunits having extensions of various lengths [7,81. In addition, it was postulated that the three basic P subunits, pSl, pS2 and pB3, have extensions from the C-terminal domains [7,9].…”

The function of Nα‐acetylation of the eye‐lens crystallins

“…Relevant fractions were lyophilized and their amino acid contents analyzed. In the experiments with the N-terminal tetrapeptides of aA,-and yII-crystallin the digests were analyzed by peptide mapping [15]. Relevant spots were subjected to amino acid analysis.…”