Identification of histidine residues involved in Zn(2+) binding to αA- and αB-crystallin by chemical modification and MALDI TOF mass spectrometry.
α-Crystallin, a member of the small heat shock protein family is the major protein of mammalian eye lens and is a molecular chaperone. As there is no protein turn over in the lens, stability of α-crystallin is one of the most crucial factors for its survival and function. We previously reported that the molecular chaperone-like activity and stability of α-crystallin dramatically increased in the presence of Zn(2+) (Biochemistry, 2008). We also reported that each subunit of α-crystallin could bind multiple zinc ions through inter-subunit bridging giving rise to enhanced stability (Biopolymers, 2011). The amino acid residues involved in zinc binding were not known. Since cysteine residues were not responsible for binding to Zn(2+), we tried to identify the histidine residues bound to zinc ions. We modified recombinant αA- and αB-crystallin with diethylpyrocarbonate (DEPC) a histidine modifying reagent, in presence and absence of Zn(2+) followed by tryptic digestion. The residues modified by DEPC were identified through peptide mass matching by MALDI mass spectrometry. We have clearly identified H79, H107 and H115 of αA-crystallin and H104, H111 and H119 of αB-crystallin as the Zn(2+) binding residues. The significance of the histidine rich sequence region of α-crystallin for its stability is discussed.
Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, 93/1 A.P.C. Road, Kolkata, 700009, India. snat14@gmail.com
Srabani
S
Karmakar
K P
KP
Das
D000595:Amino Acid Sequence; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D020539:Sequence Analysis, Protein / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D006639:Histidine; D014357:Trypsin; D015032:Zinc; D004047:Diethyl Pyrocarbonate