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A Pande, E Hanlon, J Pande; The Sulfhydryls of the Gamma Crystallins: A Vibrational Spectroscopic Analysis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4660.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the microenvironment of cysteine thiols in human and bovine γ crystallins. Methods: Recombinant bovine γB crystallin (BGB), several of its Cys to Ser mutants, and human γD crystallin (HGD) were expressed in E. coli. Raman spectra of pure recombinant proteins were measured in solution, using laser excitation at 647 nm. Results: The secondary and tertiary structures of all the mutants were nearly identical. The main difference in the Raman spectra were observed in the Cys sulfhydryl (thiol) region (2500-2800 cm-1). The thiol band profile of BGB and HGD are closely similar and consist of a doublet typical of the γ crystallins - a prominent band around 2580 cm-1 and a shoulder around 2560 cm-1. The ratio of the areas under the doublet for the two proteins (BGB/HGD) is 1.3, which is close to the ratio (1.2) of the number of cysteines in the two proteins. Thus, on average, the Raman cross-sections of the Cys residues in BGB and HGD are comparable. Subtracted spectra (recombinant wild-type BGB minus each mutant) led to the identification of thiol frequencies of some key Cys residues in BGB. Cys 15 and 22 each show two thiol bands: 2554 and 2581 cm-1 for Cys 15, and 2558 and 2576 cm-1 for Cys 22. This is consistent with the earlier x-ray crystal structure of BGB in which fractional occupancy of two different sites was observed for these two residues. Cys 18 and 78 show thiol bands at 2586 and 2581 cm-1 respectively. The Raman profiles in the thiol region for HGD suggest a weaker hydrogen bonding for the Cys residues, compared to those of BGB. Both proteins share about 80% identity in the amino acid sequence and have equivalent Cys residues at 4 positions - 18, 32, 41 and 78. Cys 109 in BGB is spatially equivalent to Cys 108 in HGD. These considerations have enabled us to assign Raman frequencies for several Cys residues in HGD. Conclusion: Raman spectra of HGD and BGB have been measured and frequency assignments made for key Cys residues. The Raman frequency of the thiol group is a direct measure of its microenvironment. These assignments will also allow us to monitor the reactivity of individual Cys residues of the proteins in solution and in the solid phase, as well as in lens tissue.
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