The N-terminal domain of alphaB-crystallin is protected from proteolysis by bound substrate.
Alpha-crystallin, a major structural protein of the lens can also function as a molecular chaperone by binding to unfolding substrate proteins. We have used a combination of limited proteolysis at low temperature, and mass spectrometry to identify the regions of alpha-crystallin directly involved in binding to the structurally compromised substrate, reduced alpha-lactalbumin. In the presence of trypsin, alpha-crystallin which had been pre-incubated with substrate showed markedly reduced proteolysis at the C-terminus compared with a control, indicating that the bound substrate restricted access of trypsin to R157, the main cleavage site. Chymotrypsin was able to cleave at residues in both the N- and C-terminal domains. In the presence of substrate, alpha-crystallin showed markedly reduced proteolysis at four sites in the N-terminal domain when compared with the control. Minor differences in cleavage were observed within the C-terminal domain suggesting that the N-terminal region of alpha-crystallin contains the major substrate interaction sites.
Aquilina
J Andrew
JA
School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. aquilina@uow.edu.au
Watt
Stephen J
SJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D038203:alpha-Crystallin B Chain; D007768:Lactalbumin; D002918:Chymotrypsin; D014357:Trypsin