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JB Smith, A Hasan, DL Smith; Identification of Regions in Human Alpha Crystallin Affected by ATP . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4797.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The chaperone-like activity of α-crystallin is enhanced by the presence of ATP. Previous studies have shown that ATP binds to α-crystallin with a Ka of 8 x 103. Characterization of this interaction using a homo-aggregate of recombinant αB-crystallin suggested a region including residues 60-122 undergoes structural modification in the presence of ATP. The extent of hydrogen-deuterium exchange at amide linkages reflects the protein structure. We have used amide hydrogen-deuterium exchange in combination with mass spectrometry to locate regions of human αA and αB in their native assemblies that change when ATP is present. Methods: Human α-crystallin, isolated by size exclusion chromatography, was incubated with 0-28 mM ATP at 37 C for 15 minutes. A 20-fold excess of D2O, also with ATP was added, and the incubation continued 30 sec. The exchange was quenched by lowering the pH to 2 and decreasing the temperature to 0 C. The amount of hydrogen exchanged for deuterium in the α-crystallins was determined by electrospray ionization mass spectrometry. The deuterated and undeuterated proteins were analyzed both as intact proteins and as peptides formed by pepsin digestion. Results: With the conditions used in this study, 82 amide hydrogens in αA and 88 hydrogens in αB were exchanged for deuterium. In the presence of ATP, the number of deuteriums incorporated was decreased, consistent with a structural change that protected some residues. The decrease in deuterium ranged from 5-12 as ATP was increased from 3.5 - 28 mM. Mass spectrometric analysis of peptides from the 7 mM ATP incubation showed the protected regions were located among residues 59-93 and and 106-127 of αA and among residues 38-54, 64-99 and 144-175 of αB. Conclusion: Hydrogen-deuterium exchange in combination with mass spectrometry yielded information indicating the interaction of ATP with native human α-crystallin causes conformational changes that make some amide linkages in αA 59-93, αA 106-127, αB 38-54, αB 64-99 and the C-terminus of αB less reactive toward hydrogen-deuterium exchange.
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