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E. E. Biswas-Fiss, D. S. Kurpad, K. Joshi, B. Sajer; Disease Mutations in the Second Extracellular Loop of the Human Retina Specific ABC Transporter, ABCA4, Impart Structural Defects in This Domain. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1093.
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The retina specific ABC transporter, ABCA4, is associated with a broad range of inherited macular degenerations including Stargardt disease (STDG), autosomal recessive cone rod dystrophy (arCRD) and fundus flavimaculatus (FFM). A unique topological feature of the ABCA family of ABC transporters is the presence of long extracellular (EC) loops or domains, sequences of which are highly conserved. A cluster of point mutations in the second extracellular domain (ECD2) has been identified in patients with the aforementioned degenerative diseases.
We have analyzed the solution structure of this domain by circular dichroism (CD) spectroscopy and investigated the structural changes upon single, disease- associated point mutations.
CD analysis of purified, recombinant forms of this protein demonstrated that the ECD2 domain has a highly ordered and stable structure comprised of 27±3% alpha helix, 20±3% beta-pleated sheet, and 53±3% coil. Significant changes in the CD spectra were observed in disease mutants as well as in their predicted protein conformations. Thermal melting by CD spectroscopic analysis demonstrated alteration in the overall stability of the structures of ECD2 upon mutation. The point mutations also altered the environment in the core of the protein structure as observed through measurement of intrinsic tryptophan fluorescence (EX 295 nm and EM of 320-500 nm).
These results support the hypothesis that the extracellular domains of ABCA4 play an important role in its transport function which is likely dependent upon specific conformational features of these domains; furthermore Stargardt disease associated mutations may lead to significant changes in the structural features of these domains.
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