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J. Pande, A. Pande; The Cataract-Linked R14C Mutant of Human Gamma-D Crystallin Has an Anomalous Cys14 pKa Which Facilitates the Ready Formation of Intermolecular Disulfide Crosslinks. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5026. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The R14C mutant of human Gamma-D crystallin (HGD) has been shown to rapidly form disulfide-crosslinked oligomers that lead to light scattering and opacity in solution, while maintaining a native-like protein conformation and stability (1). In order to determine the nature of the newly introduced cysteine residue in R14C that could explain its ready oligomerization, we have examined recombinant wild-type HGD and R14C by Raman spectroscopy. This technique is uniquely suited to probe the environment of the thiol and disulfide groups of Cys residues.
Recombinant HGD and R14C were expressed in E.coli and purified from the soluble fractions using size-exclusion and cation-exchange chromatography (1). Raman spectra of solutions of HGD and R14C at protein concentrations around 4-6 mM were measured at pH 7 and 4.5 using a Renishaw inVia Raman microscope with a 785 nm laser excitation.
In the spectral region 500-550 cm-1, typical of-S-S- frequencies, R14C shows a number of bands -predominantly around 510, 520 and 540 cm-1 - in sharp contrast to HGD which has no detectable -S-S- bands. These modes correspond to the gauche-gauche-gauche, gauche-gauche trans and trans-gauche-trans conformations observed in inter-protein disulfide crosslinks. Surprisingly, the spread of multiple -S-S- frequencies appears not only at pH 7 but also at pH 4.5 in the mutant.
The appearance of multiple disulfide frequencies in the Raman spectrum of R14C suggests that this mutant is able to form a number of conformational isomers of the intermolecular disulfide crosslinks. Furthermore, the presence of -S-S- crosslinks at pH 4.5 clearly suggests that Cys14 is ionized even at a low pH, and therefore has an anomalously low pKa value.(1) Pande, A., Pande, J., Asherie, N., Lomakin, A., Ogun, O., King, J.A., Lubsen, N.H., Walton, D. and Benedek, G.B. (2000) Proc. Natl. Acad. Sci. U.S.A., 97, 1993-1998.
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