Purchase this article with an account.
EE Biswas, SB Biswas; Structure-Function Analysis of Macular Dystrophy Associated Mutations Influencing the Second Nucelotide Binding Domain of the Human Retinal ABC Transporter . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1401.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The retina specific ABC transporter has been linked through human genetic studies to a variety of inherited retinal dystrophies, including Stargardt disease, cone-rod dystrophy, fundus flavimaculatus and age related macular degeneration. Our goal has been to examine the functional consequences of several disease-associated missense mutations on the structure and function of second nucleotide binding domain of ABCR, and correlate these observed changes to disease pathogenesis. Methods: The second nucleotide-binding domain of ABCR is defined as amino acids 1898-2273 (bp 5733-6900). The domain was cloned into a T7 expression system vector which was then used as a template to create constructs harboring the following disease associated mutations: G1961E, L1971R, L2027F, and D2177N. The wild type and mutant proteins were expressed and purified to homogeneity. Using fluorescence based methods the structure and energetics of ATP binding of NBD2 were analyzed and correlated with the kinetics of ATP hydrolysis. Results: The KD and Δ;G0 values of nucleotide binding were determined for wild type and mutant proteins using fluorescence polarization. In all mutants examined, the binding constants for mutant proteins were less than that observed for the wild type protein. Correlation of the binding parameters with rate of ATP hydrolysis (Vmax) demonstrated two separate types of effects. In most cases, the decrease in binding affinity was associated with a significant increase in Vmax. However, in the case of mutant L2027F, a small decrease in Vmax was observed. Conclusion: The data suggest that in some instances there appears to be an underlying attempt to compensate in order to maintain overall function in response to certain mutations. Structural differences may underlie the overall effects on ATP hydrolysis observed with some of the mutations. Homology based modeling studies are currently underway to address these issues. CR-None
This PDF is available to Subscribers Only