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Identification of Histidine Residues Involved in Zn2+ Binding to αA- and αB-Crystallin by Chemical Modification and MALDI TOF Mass Spectrometry

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Abstract

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 Zn2+ (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 Zn2+, 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 Zn2+ 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 Zn2+ binding residues. The significance of the histidine rich sequence region of α-crystallin for its stability is discussed.

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Abbreviations

ACN:

Acetonitrile

DEPC:

Diethylpyrocarbonate

DHAP:

2,5-Dihydroxy acetophenon

HCCA:

α-Cyano-4-hydroxycinnamic acid

IPTG:

Isopropyl-β-d-thiogalactopyranoside

MALDI:

Matrix assisted laser desorption ionization

MS:

Mass spectroscopy

TFA:

Trifluoroacetic acid

TOF:

Time of flight

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Acknowledgments

This work was supported by a CSIR grant (No. 37/1218/05/EMR-II to K.P.D.). MALDI facility at our Institute was supported by a DST-IRHPA grant. We would like to acknowledge Mr. Dipak Chandra Konar for his assistance in purifying the recombinant αA-and αB-crystallin. Thanks are due to Mr. J. Guin for help with the MALDI experiments. S.K. was supported in part by a senior research fellowship from Bose Institute.

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  1. Srabani Karmakar

    Present address: Department of Chemical Sciences, Tata Institute of Fundamental Research, 1 Homi Bhabha Road, Navy Nagar, Colaba, Mumbai, 400005, India

Authors and Affiliations

  1. Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, 93/1 A.P.C. Road, Kolkata, 700009, India

    Srabani Karmakar & K. P. Das

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  1. Srabani Karmakar
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  2. K. P. Das
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Correspondence to K. P. Das.

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Karmakar, S., Das, K.P. Identification of Histidine Residues Involved in Zn2+ Binding to αA- and αB-Crystallin by Chemical Modification and MALDI TOF Mass Spectrometry. Protein J 31, 623–640 (2012). https://doi.org/10.1007/s10930-012-9439-0

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