Purchase this article with an account.
H.A. Koteiche, H.S. Mchaourab; Cataract-linked Mutants of A-crystallin: Chaperones or "Unfoldases"? . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2141.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: A number of point mutations in αA-crystallin have been genetically linked to autosomal dominant cataract. The detailed molecular mechanism underlying this linkage remains unclear. In this study, we explore the consequences of two mutations, R49C and R116C, on the recognition and binding of non-native states of T4 Lysozyme (T4L) by αA-crystallin. Methods: A set of destabilized T4L mutants with free energy of unfolding in the 5-10 kcal/mol range was constructed, expressed and purified. Each of the mutants contains a cysteine at a non-destabilizing site. The purified mutants were reacted with either a fluorescent or a paramagnetic reporter group. Electron Paramagnetic Resonance (EPR) or Fluorescence spectroscopies were employed to directly observe the formation of the complex between labeled T4L mutants and αA-crystallin. To obtain binding isotherms, αA and its variants were incubated with T4L mutants at various molar ratios and the bound T4L fraction determined by EPR spectroscopy. Results: Both αA-crystallin mutants bind destabilized T4L mutants to a higher extent than the WT. In the context of the two-mode binding mechanism of the α-crystallins, the origin of the increased binding appears to be an enhanced affinity of the high capacity mode. The activation of this mode is more significant in R116C as revealed by a more defined curvature in its binding isotherm. Analysis of the αA/T4L complex by fluorescence spectroscopy directly confirms that T4L has enhanced access to the high capacity sites in the αA mutants. Because the high capacity mode selectively binds globally unfolded states, the effect of the mutations is to enhance the affinity of αA-crystallin to these states. Conclusions: It has been proposed (1) that mutations in αA-crystallin, associated with congenital cataracts, lead to a gain of function. The results of this study suggest that such gain of function might be related to an increased affinity to the unfolded states of stable proteins. Thus, the deleterious effect of the mutations is mediated by a new "unfoldase" activity of αA-crystallin as a result of thermodynamic coupling to the folding reaction of the substrate. (1) B.A. Cobb and J.M. Petrash (2000). Biochemistry 39, 15791-8.
This PDF is available to Subscribers Only