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K.J. Lampi, M. Harms, D.M. Kapfer; Deamidation at Asparagine 157 in Human betaB1-crystallin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2358.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To characterize an in vivo site of deamidation identified at asparagine 157 in human betaB1-crystallin (Hanson et al., Exp. Eye Res. 71, 195-207, 2000). Methods: Wildtype and N157D betaB1-crystallins were recombinantly expressed and purified. Secondary structure was determined by far UV-circular dichroism, and relative stability compared by thermal denaturation. Oligomer size was predicted from molar mass determined by size exclusion chromatography in line with multi-angle laser light scattering. Results: Wild type and a previously characterized deamidated betaB1, Q204E, eluted on cation exchange chromatography between 100-150 mM NaCl, while N157D did not bind to the cation column (SP Sepharose Fast Flow, Amersham Pharmacia). N157D was purified from e. coli proteins using anion exchange chromatography and was immunoreative against a rat beta-crystallin antibody. N157D had a minimum in ellipticity at 205 nm and more closely matched the circular dichroism spectrum of wild type than Q204E. After heating at 55 degrees Celsius for 730 minutes, the majority of N157D remained soluble, similar to what was seen for wild type. This is in contrast to Q204E, which precipitated upon heating. Conclusions: Differences between N157D and Q204E are most likely due to their different sites in the protein structure. Glutamine 204 is located at the interface between two interacting domains of partner subunits and is predicted to destabilize the dimer. Asparagine 157, while also located in the C-terminal domain, is oriented away from the surface of the dimer. Future studies will determine the relative stabilities of other in vivo sites of deamidation.
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