title: Local microdomain structure in the terminal extensions of betaA3- and betaB2-crystallins.
abstract: OBJECTIVE
  Although the crystal structures of the core domains of bovine betaB2-crystallin have been determined and those of other betagamma-crystallins modeled, the positions of the N- and C-termini are not resolvable by X-ray crystallography. Here we model the possible structural organization of the terminal arms of mouse betaA3- and betaB2-crystallins and test this model against the results of partial proteolysis.
  
  
  METHODS
  The secondary structure of the terminal extensions was predicted by 3 different methods, one a nearest-neighbor method modified to use overlapping sequence tripeptides. Recombinant betaA3- and betaB2-crystallins were expressed using baculovirus vectors in S. frugiperda Sf9 cells. Crystallins were sequenced by the Edman degradation method.
  
  
  RESULTS
  The N-terminal extension of betaB2-crystallin includes a series of hydrophilic residues from Q-11 to Q-9 which have high propensity of a helical conformation. The N-terminal arm of betaA3-crystallin is also predicted to have two helical segments, from Q-24 to E-20 and M-13 to A-12. Partial characterization of the baculovirus extract showed a thiol protease inhibited by leupeptin and E-64. As predicted by the model, recombinant betaB2-crystallin subjected to partial proteolysis was cleaved adjacent to the helical domain, while the N-terminal cleavage site in recombinant betaA3-crystallin was within 1 residue of an interhelical junction. Our model also predicts the products of partial proteolytic degradation of betaB2- and betaA3-crystallins from human, rat, bovine and chicken lenses incubated with the protease m-calpain.
  
  
  CONCLUSIONS
  These results suggest the existence of local microdomain structures in the N- and C-terminal extensions of betaA3- and betaB2-crystallins, which appear to be more susceptible to proteolytic degradation in regions adjacent to these putative domains.
authors.affiliation: National Eye Institute, NIH, Bethesda, MD 20892-1860, USA. sergeev@helix.nih.gov
authors.forename: Y V
authors.initials: YV
authors.lastname: Sergeev
authors.forename: L L
authors.initials: LL
authors.lastname: David
authors.forename: H C
authors.initials: HC
authors.lastname: Chen
authors.forename: J N
authors.initials: JN
authors.lastname: Hope
authors.forename: J F
authors.initials: JF
authors.lastname: Hejtmancik
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002417:Cattle; D002645:Chickens; D003459:Crystallins / Q000737:chemistry* / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D008433:Mathematics; D051379:Mice / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D017421:Sequence Analysis; D014162:Transfection
chemical_list: D003459:Crystallins; D011480:Protease Inhibitors; D011994:Recombinant Proteins