Structure, stability, and chaperone function of αA‐crystallin: Role of N‐terminal region

Abstract

Small heat shock protein alphaA-crystallin, the major protein of the eye lens, is a molecular chaperone. It consists of a highly conserved central domain flanked by the N-terminal and C-terminal regions. In this article we studied the role of the N-terminal domain in the structure and chaperone function of alphaA-crystallin. Using site directed truncation we raised several deletion mutants of alphaA-crystallin and their protein products were expressed in Escherichia coli. Size exclusion chromatography of these purified proteins showed that deletion from the N-terminal beyond the first 20 residues drastically reduced the oligomeric association of alphaA-crystallin and its complete removal resulted in a tetramer. Chaperone activity of alphaA-crystallin, determined by thermal and nonthermal aggregation and refolding assay, decreased with increasing length of deletion and little activity was observed for the tetramer. However it was revealed that N-terminal regions were not responsible for specific recognition of natural substrates and that low affinity substrate binding sites existed in other part of the molecule. The number of exposed hydrophobic sites and the affinity of binding hydrophobic probe bis-ANS as well as protein substrates decreased with N-terminal deletion. The stability of the mutant proteins decreased with increase in the length of deletion. The role of thermodynamic stability, oligomeric size, and surface hydrophobicity in chaperone function is discussed. Detailed analysis showed that the most important role of N-terminal region is to control the oligomerization, which is crucial for the stability and in vivo survival of this protein molecule.

Roles of the N‐ and C‐terminal sequences in Hsp27 self‐association and chaperone activity

“…This study is an effort toward that goal that is based on correlating the oligomeric state of the protein with its chaperone activity and subunit-exchange kinetics to define the functional contributions of the N-and C-sequences of the protein. The importance of these regions of small heat shock proteins has been widely recognized and studied in both metazoan 27,28,[37][38][39][40][41][42][43][44][45][46][47][48] and bacterial heat shock proteins. 35,49,50 This study examines the mechanistic role of the N-and C-terminal sequences of human Hsp27 in the temperature-dependent self-association and in the chaperone activity of the protein.…”

“…Similar trends have been reported for aA-crystallin in that N-terminal deletion variants exhibit slower Lelj-Garolla and Mauk exchange rates than observed for the wild-type protein, and N-terminal truncation variants from which >50 amino acid residues were deleted showed no chaperone activity, no oligomerization greater than tetramers and no subunit exchange. 37 With sufficiently slow subunit exchange (k off 10 À5 sec…”

A S52P mutation in the ‘α‐crystallin domain’ of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function

“…were obtained from Sisco Research Laboratories (Mumbai, India). Bovine b L -crystallin was purified from bovine eye lenses, as described previously [68]. All other chemicals were of analytical grade.…”

“…The chaperone activity was determined with two client proteins: insulin and b L -crystallin as described previously [6,68]. Both assays were performed with the aid of UV spectrophotometer (Perkin Elmer, Boston, MA, USA).…”

Molecular characterization of rat cvHsp/HspB7 in vitro and its dynamic molecular architecture

“…generally, the vibronic transition beyond 285 nm arises from Trp residues, whereas the bands between 285 and 275 nm reflect the molecular packing of Trp and Tyr residue side chains (18). The cvHsp does not contain Trp residue in its sequence, which suggests that the subtle molecular packing-induced alterations were contributed by Tyr residue side chains.…”