IERG Abstracrt Book.indd - LV Prasad Eye Institute

Paper Session I, Molecular mechanisms of eye diseases,July 31, 2010, 9.20 -11.00 hrsOral Presentation29Chairs: G Kumarmanickavel and Subhabrata ChakrabartiIPT 001Evolution of Ca2+-Mediated Stability in Diverse bg-Crystallin DomainsAmita Mishra, Shashi Kumar Suman, Yogendra SharmaCentre for Cellular and Molecular Biology, Hyderabad, IndiaPurpose: Topologically similar bg-crystallin fold, found in all three kingdoms, appears to bean example of natures’ extreme engineering designed for Ca2+-mediated domain stability,though its molecular basis is not known and present an evolutionary paradox. Our goal was tounderstand how does the Ca2+-dependent generic gain in stability was evolved by differentiallydesigned domains.Methods: Crystallin domains from various genomes were selected, cloned and overexpressed.We performed the equilibrium unfolding of more than 12 structurally similar, single bg-crystallindomains with canonical Ca2+-binding motif i.e., N/DN/DXXS/TS sequence.Results: We report that these structurally similar domains are differentially stabilized by Ca2+.While some domains (flavollin and vibrillin), remain unaffected, others (clostrillin) undergomoderate stabilization by Ca2+ (Δc1/2 ~0.5 M). On the other hand, centillin gains very highstability after binding Ca2+ (Δc1/2 >1 M). We have identified causal residues using selectivemutations that do not disable the Ca2+-binding but cause increase or decrease the gain instability by Ca2+. Even homologous mutations (Thr/Ser) alter the gain in stability. A polar orhydrophobic residue at 3rd position nullifies the Ca2+-dependent stabilization. Presence ofpolar (1st) residue, involved in indirect coordination via water in centillin (F75D), enhances thestability significantly, probably due to solvent displacement. The results demonstrate how thenature of a residue in the N/DN/DXXS/TS motif governs the Ca2+-dependent gain or loss instability. Such a phenomenon is implicated in the Ca2+-dependent protection of spores andspherule using Protein S and spherulin 3a.Conclusions: Our analysis generates the possibilty of designing a protein with ultra highstability (in apo form) by calculated combinations at the binding site. We demonstrate the basisof differential Ca2+-dependent stability in various proteins with bg-crystallin domains and itsdesign route taken during evolution depending on the function of the domain in the respectiveorganisms.IPT 002Cross-Talk between the cAMP-PKA and RhoA-Rho Kinase Signaling Pathways inTrabecular Meshwork CellsCharanya Ramachandran, 1 Sangly P Srinivas 21Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, L V Prasad Eye Institute, Hyderabad,India, 2 School of Optometry, Indiana University, Bloomington, Indiana, USA