ATP and the core "alpha-crystallin" domain of the small heat-shock proteinalpha B-crystallin

Electrospray ionization mass spectrometry (ESI-LC/MS) of tryptic digests of human alpha B-crystallin in the presence and absence of ATP identified fou r residues located within the core "alpha-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 pl ace of trypsin. The chymotryptic fragments of human alpha B-crystallin prod uced in the presence and absence of ATP were analyzed using liquid chromato graphy-tandem mass spectrometry (LC-MS/MS), Seven chymotryptic cleavage sit es, Trp(60), Phe(61), Phe(75), Phe(84), Phe(113), Phe(118), and Tyr(122), l ocated near or within the core alpha-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analo gs of ATP were less protective than ATP against proteolysis by trypsin or c hymotrypsin. ATP had no effect on the enzymatic: activity of trypsin and th e K-m for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the a bsence of ATP. The results demonstrated an ATP-de; pendent structural modif ication in the Gore alpha-crystallin domain conserved in nearly all identif ied small heat-shock proteins that act as molecular chaperones.