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Interaction of Cu+2 with α-Crystallin: A Biophysical and Mass Spectrometric Study

[ Vol. 25 , Issue. 3 ]


Srabani Karmakar* and K.P. Das   Pages 275 - 284 ( 10 )


Background: αA- and αB- crystallin are members of small heat shock protein family with chaperone property. Their interactions with Cu2+ ions are reported in neurodegenerative diseases. We have been studying the effect of small ionic molecules on the stability of α-crystallin. Cu2+ is co-ordinated with αB-crystallin involving three histidine residues and one aspartic acid residue as potential binding sites. However, copper binding sites for the oligomeric native protein ╬▒A-crystallin protein is not known.

Objective: The objective of this study was to study oligomerization and stability of αA- and ╬▒Bcrystallin in presence and absence of Cu2+ ions and to find binding sites of Cu2+ on αA-crystallin.

Methods: The recombinant Human αA- and αB-crystallin proteins were purified after overexpression from the E. coli BL21DE3 cell lysate by a combination of ion-exchange and gel filtration chromatography. Mass analysis of αA- and αB-crystallin in absence and presence of Cu2+ were carried out by MALDI TOF MS. Stability of αA-crystallin in presence and absence of Cu2+ was determined by equilibrium urea denaturation experiments. The equilibrium urea unfolding profiles of the αA-crystallin in absence and presence of different Cu2+ concentrations were fitted according to the three state model of protein unfolding. Dynamic Light Scattering (DLS) measurements were carried out to detect the oligomeric size of αA-crystallin in presence and absence of Cu2+ during urea unfolding. Histidine residues were modified by DEPC (Diethyl pyro carbonate). Chemically modified and unmodified αA-crystallin was digested by trypsin prior to MALDI MS analysis. Cu2+ pre-incubation was done before the chemical modification.

Results: Mass spectrometric detection of intact protein allows direct measurement of Cu2+ ions bound to the protein. Thus the average numbers of Cu2+ bound to αA- and αB-crystallin were 4.2 and 3.6 respectively per subunit. It is seen that in presence of Cu2+ ions the free energy (ΔG) of unfolding of αA-crystallin almost doubled. The size analysis by dynamic light scattering data clearly indicated that in presence of Cu2+ ions the oligomeric size remain unchanged with increasing urea solutions. Mass spectrometric detection with chemical modification of histidine residues of αA-crystallin in presence and absence of Cu2+ indicated that amino acid residues H107, H100, H115 of αA-crystallin are involved in Cu2+ binding.

Conclusion: Our results indicated that Cu2+ helped in increasing stability of αA-crystallin and three histidine residues H100, H107 and H115 of αA-crystallin are Cu2+ binding residues.


α-crystallin chaperone, stabilization by Cu2+, dynamic light scattering of α-crystallin, urea unfolding of α- crystallin, MALDI TOF mass spectrometry, histidine residues.


Department of Biotechnology, Techno India University West Bengal, EM-4 Sector V, Salt Lake, Kolkata-700091, Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, 93/1 A.P.C. Road, Kolkata-700009

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