title: ATP and the core "alpha-Crystallin" domain of the small heat-shock protein alphaB-crystallin.
abstract: Electrospray ionization mass spectrometry (ESI-LC/MS) of tryptic digests of human alphaB-crystallin in the presence and absence of ATP identified four 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 place of trypsin. The chymotryptic fragments of human alphaB-crystallin produced in the presence and absence of ATP were analyzed using liquid 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 alpha-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 alpha-crystallin domain conserved in nearly all identified small heat-shock proteins that act as molecular chaperones.
authors.affiliation: Department of Biological Structure, University of Washington, Seattle, Washington 98195-7420, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Muchowski
authors.forename: L G
authors.initials: LG
authors.lastname: Hays
authors.forename: J R
authors.initials: JR
authors.lastname: Yates
authors.forename: J I
authors.initials: JI
authors.lastname: Clark
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D002853:Chromatography, Liquid; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D014357:Trypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D003459:Crystallins; D000255:Adenosine Triphosphate; D014357:Trypsin