Structure and interactions in α-crystallin probed through thiol group reactivity


a-Crystallin is the major structural protein of eye lens of vertebrates. In human lens, the ratio of aA-crystallin to aB-crystallin was found to be 3:1. aA-Crystallin contains two cysteine residues at positions 131 and 142, which are at the junction between the a-crystallin domain and the C-terminal tail. We used the accessibility of the thiol groups by Ellman’s reagent (DTNB) as a tool to gain information about the various structural perturbations of hinge region of a-crystallin and during the binding with substrates. In the native condition, the cys-142 though reacted quite fast was not fully exposed. Several reagents were used to see the accessibility of cys-131. Rate constant for cys-131 was increased gradually with increase in the concentration of reagents. The bindings of substrates are affected by the accessibility of thiol indicating that the substrates bind to the hinge region of a-crystallin. By blocking of cys-142, it was observed that the accessibility of one thiol depends on the other thiol, and they are not independent. The hinge region of a-crystallin is very important as substrate binding site and from this study we have got various structural information about that region.

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Saha, S. and Das, K. (2013) Structure and interactions in α-crystallin probed through thiol group reactivity. Advances in Biological Chemistry, 3, 427-439. doi: 10.4236/abc.2013.35046.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Das, K.P. and Surewicz, W.K. (1995) Temperature-induced exposure of hydrophobic surfaces and its effect on the chaperone activity of alpha-crystallin. FEBS Letters, 369, 321-325.
[2] Biswas, A., Saha, S. and Das, K.P. (2002) Structural features of molecular chaperones: A possible micellar connection. Journal of Surface Science and Technology, 18, 1-24.
[3] Saha, S. and Das, K.P. (2007) Unfolding and refolding of bovine alpha-crystallin in urea and its chaperone activity. The Protein Journal, 26, 315-326.
[4] Saha, S. and Das, K.P. (2004) Relationship between chaperone activity and oligomeric size of recombinant human alphaA- and alphaB-crystallin: A tryptic digestion study. Proteins, 57, 610-617.
[5] Narberhaus, F. (2002) Alpha-crystallin-type heat shock proteins: Socializing minichaperones in the context of a multi-chaperone network. Microbiology and Molecular Biology Reviews, 66, 64-93.
[6] Sharma, K.K., Kaur, H., Kumar, G.S. and Kester, K. (1998) Interaction of 1,1’-bi(4-anilino)naphthalene-5,5’- disulfonic acid with alpha-crystallin. The Journal of Biological Chemistry, 273, 8965-8970.
[7] Sharma, K.K., Kumar, G.S., Murphy, A.S. and Kester, K. (1998) Identification of 1,1’-bi(4-anilino)naphthalene- 5,5’-disulfonic acid binding sequences in alpha-crystallin. The Journal of Biological Chemistry, 273, 15474-15478.
[8] Datta, S.A. and Rao, C.M. (2000) Packing-induced conformational and functional changes in the subunits of alpha-crystallin. The Journal of Biological Chemistry, 275, 41004-41010.
[9] Pasta, S.Y., Raman, B., Ramakrishna, T. and Rao, C.M. (2002) Role of the C-terminal extensions of alpha-crystallins. Swapping the C-terminal extension of alpha-crystallin to alphaB-crystallin results in enhanced chaperone activity. The Journal of Biological Chemistry, 277, 45821-45828.
[10] Santhoshkumar, P. and Sharma, K.K. (2001) Phe71 is essential for chaperone-like function in alpha A-crystallin. The Journal of Biological Chemistry, 276, 47094-47099.
[11] Sharma, K.K., Kaur, H. and Kester, K. (1997) Functional elements in molecular chaperone alpha-crystallin: Identification of binding sites in alpha B-crystallin. Biochemical and Biophysical Research Communications, 239, 217-222.
[12] Sharma, K.K., Kumar, R.S., Kumar, G.S. and Quinn, P.T. (2000) Synthesis and characterization of a peptide identified as a functional element in alphaA-crystallin. The Journal of Biological Chemistry, 275, 3767-3771.
[13] Bova, M.P., Mchaourab, H.S., Han, Y. and Fung, B.K. (2000) Subunit exchange of small heat shock proteins. Analysis of oligomer formation of alphaA-crystallin and Hsp27 by fluorescence resonance energy transfer and site-directed truncations. The Journal of Biological Chemistry, 275, 1035-1042.
[14] Feil, I.K., Malfois, M., Hendle, J., van der Zandt H. and Svergun, D.I. (2001) A novel quaternary structure of the dimeric alpha-crystallin domain with chaperone-like activity. The Journal of Biological Chemistry, 276, 12024-12029.
[15] Kokke, B.P.A., Leroux, M.R., Candido, E.P.M., Boelens, W.C. and de Jong, W.W. (1998) Caenorhabditis elegans small heat-shock proteins Hsp12.2 and Hsp12.3 form tetramers and have no chaperone-like activity. FEBS Letters, 433, 228-232.
[16] Leroux, M.R., Ma, B.J., Batelier, G., Melki, R. and Candido, E.P.M. (1997) Unique structural features of a novel class of small heat shock proteins. The Journal of Biological Chemistry, 272, 12847-12853.
[17] Das, K.P., Petrash, J.M. and Surewicz, W.K. (1996) Conformational properties of substrate proteins bound to a molecular chaperone alpha-crystallin. The Journal of Biological Chemistry, 271, 10449-10452.
[18] Lakowicz, J.R. (1983) Principles of fluorescence spectroscopy. Plenum Press, New York.
[19] Glazer, A.N. (1970) Specific chemical modification of proteins. Annual Review of Biochemistry, 39, 101.
[20] Means, G.E. and Feeney, R.E. (1971) Chemical modifications of proteins. Holden-Day Sanfransisco California.
[21] Horwitz, J., Huang, Q.L., Ding, L.L. and Bova, M.P. (1998) Lens alpha-crystallin: Chaperone-like properties. Methods in Enzymology, 290, 365-383.
[22] Bhattacharyya, J. and Das, K.P. (1999) Effect of sur-fac- tants on the prevention of protein aggregation during unfolding and refolding processes—Comparison with molecular chaperone-crystallin. Journal of Dispersion Science and Technology, 20, 1163-1178.
[23] Muchowski, P.J. and Clark, J.I. (1998) ATP-enhanced molecular chaperone functions of the small heat shock protein human alphaB crystalline. Proceedings of the National Academy of Sciences, 95, 1004-1009.
[24] Palmisano, D.V., Groth-Vasselli, B., Farnsworth, P.N. and Reddy, M.C. (1995) Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy. Biochimica et Biophysica Acta, 1246, 91-97.
[25] Rawat, U. and Rao, M.J. (1998) Interactions of chaperone alpha-crystallin with the molten globule state of xylose reductase. Implications for reconstitution of the active enzyme. The Journal of Biological Chemistry, 273, 9415-9423.
[26] Wang, K. and Spector, A. (2000) alpha-crystallin prevents irreversible protein denaturation and acts cooperatively with other heat-shock proteins to renature the stabilized partially denatured protein in an ATP-dependent manner. European Journal of Biochemistry, 267, 4705-4712.
[27] Wang, K. and Spector, A. (2001) ATP causes small heat shock proteins to release denatured protein. European Journal of Biochemistry, 268, 6335-6345.
[28] Biswas, A. and Das, K.P. (2004) Role of ATP on the interaction of alpha-crystallin with its substrates and its implications for the molecular chaperone function. The Journal of Biological Chemistry, 279, 42648-42657.
[29] Hasan, A., Smith, D.L. and Smith, J.B. (2002) Alpha-crystallin regions affected by adenosine 5’-triphosphate identified by hydrogen-deuterium exchange. Biochemistry, 41, 15876-15882.
[30] Kumar, M.S., Mrudula, T., Mitra, N. and Reddy, G.B. (2004) Enhanced degradation and decreased stability of eye lens alpha-crystallin upon methylglyoxal modification. Experimental Eye Research, 79, 577-583.
[31] Augusteyn, R.C., Hum, T.P., Putilin, T.P. and Thomson, J.A. (1987) The location of sulphydryl groups in alpha-crystallin. Biochimica et Biophysica Acta, 915, 132-139.
[32] Siezen, R.J., Coenders, F.G. and Hoenders, H.J. (1978) Three classes of sulfhydryl group in bovine alpha-crystallin according to reactivity to various reagents. Biochimica et Biophysica Acta, 537, 456-465.
[33] Doss-Pepe, E.W., Carew, E.L. and Koretz, J.F. (1998) Studies of the denaturation patterns of bovine alpha-crystallin using an ionic denaturant, guanidine hydrochlo-ride and a non-ionic denaturant, urea. Experimental Eye Research, 67, 657-679.
[34] Sun, T.X., Akhtar, N.J. and Liang, J.J.N. (1999) Thermodynamic stability of human lens recombinant alphaA- and alphaB-crystallins. The Journal of Biological Chemistry, 274, 34067-34071.
[35] Biswas, A. and Das, K.P. (2004) SDS induced structural changes in alpha-crystallin and its effect on refolding. The Protein Journal, 23, 529-538.
[36] Pasta, S.Y., Raman, B., Ramakrishna, T. and Rao, C.M. (2004) The IXI/V motif in the C-terminal extension of alpha-crystallins: Alternative interactions and oligomeric assemblies. Molecular Vision, 10, 655-662.
[37] Kim, K.K., Kim, R. and Kim, S.H. (1998) Crystal structure of a small heat-shock protein. Nature, 394, 595-599.
[38] van Montfort, R.L., Basha, E., Friedrich, K.L., Slingsby, C. and Vierling, E. (2001) Crystal structure and assembly of a eukaryotic small heat shock protein. Nature Structural Biology, 8, 1025-1030.
[39] Kontorow, M., Horwitz, J., van Boekel, M.A.M., de Jong W.W. and Piatigorsky, J. (1995) Conversion from oligomers to tetramers enhances autophosphorylation by lens alpha A-crystallin. Specificity between alpha A- and alpha B-crystallin subunits. The Journal of Biological Chemistry, 270, 17215-17220.
[40] Rajan, R. and Balaram, P. (1996) A model for the interaction of trifluoroethanol with peptides and proteins. International Journal of Peptide and Protein Research, 48, 328-336.
[41] Srinivas, V., Santhoshkumar, P. and Sharma, K.K. (2002) Effect of trifluoroethanol on the structural and functional properties of alpha-crystallin. Journal of Protein Chemistry, 21, 87-95.
[42] Horwitz, J. (2003) Alpha-crystallin. Experimental Eye Research, 76, 145-153.
[43] Muchowski, P.J., Hays, L.G., Yates, J.R. and Clark 3rd, J.I. (1999) ATP and the core “alpha-crystallin” domain of the small heat-shock protein alphaB-crystallin. The Journal of Biological Chemistry, 274, 30190-30195.
[44] Raman, B., Ramakrishna, T. and Rao, C.M. (1995) Temperature dependent chaperone-like activity of alpha-crystallin. FEBS Letters, 365, 133-136.
[45] Reddy, G.B., Das, K.P., Petrash, J.M. and Surewicz, W.K. (2000) Temperature-dependent chaperone activity and structural properties of human alphaA- and alphaB-crystallins. The Journal of Biological Chemistry, 275, 4565-4570.
[46] Maiti, M., Kono, M. and Chakraborti, B. (1988) Heat-induced changes in the conformation of alpha- and beta-crystallins: Unique thermal stability of alpha-crystallin. FEBS Letters, 236, 109-114.

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