ATP and the Core "α-Crystallin" Domain of the Small Heat-shock Protein αB-crystallin

Paul J. Muchowski, Lara G. Hays, John R. Yates, John I. Clark
1999 Journal of Biological Chemistry  
Electrospray ionization mass spectrometry (ESI-LC/ MS) of tryptic digests of human ␣B-crystallin in the presence and absence of ATP identified four residues located within the core "␣-crystallin" domain, Lys 82 , Lys 103 , Arg 116 , and Arg 123 , that were shielded from the action of trypsin in the presence of ATP. In control experiments, chymotrypsin was used in place of trypsin. The chymotryptic fragments of human ␣B-crystallin produced in the presence and absence of ATP were analyzed using
more » ... quid chromatography-tandem mass spectrometry (LC-MS/MS). Seven chymotryptic cleavage sites, Trp 60 , Phe 61 , Phe 75 , Phe 84 , Phe 113 , Phe 118 , and Tyr 122 , located near or within the core ␣-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analogs of ATP were less protective than ATP against proteolysis by trypsin or chymotrypsin. ATP had no effect on the enzymatic activity of trypsin and the K m for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the absence of ATP. The results demonstrated an ATP-dependent structural modification in the core ␣-crystallin domain conserved in nearly all identified small heatshock proteins that act as molecular chaperones.
doi:10.1074/jbc.274.42.30190 pmid:10514509 fatcat:e7o4zll4w5bs5gcpnvekm2yusm