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K. Runager, J. Underhaug, R. V. Basaiawmoit, Z. Valnickova, T. Kristensen, D. E. Otzen, G. K. Klintworth, N. C. Nielsen, J. J. Enghild; Naturally Occurring Mutations Alter the Structure and Stability of the Fourth Fasciclin Domain of the Transforming Growth Factor Beta Induced Protein (TGFBIp). Invest. Ophthalmol. Vis. Sci. 2008;49(13):1016. doi: https://doi.org/.
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Mutations in the transforming growth factor beta induced protein (TGFBIp) have been linked to a number of corneal dystrophies. However, little is known about the composition of the protein aggregates found in these diseases as well as the mechanism of aggregation. This study addresses these questions by studying the structural and biophysical effects of mutations in the fourth fasciclin domain (FAS4) of TGFBIp.
Wild-type and mutants of FAS4 were cloned in the pET SUMO vector (Invitrogen) and expressed in E. coli in either Luria-Bertani (LB) medium for biophysical studies or M9 minimal medium supplemented with 15N labeled ammonium chloride and 13C labeled glucose for nuclear magnetic resonance (NMR) spectroscopy. Protein was purified as described in the SUMO expression protocol. FAS4 stability was analyzed by 0-8 M urea transverse urea gels and circular dichroism (CD) spectroscopy using a Jasco J-810 spectropolarimeter. All NMR spectra were acquired at 300K on a Bruker 700 MHz ultra-shielded wide bore magnet.
The biophysical study of FAS4 revealed that single amino acid substitutions alter the stability significantly. The A546T mutation causing a variant of lattice corneal dystrophy (LCD) showed a decreased stability compared to the wild-type whereas the R555W mutation causing granular corneal dystrophy (GCD) type I was significantly more thermodynamically stable than the wild-type. NMR spectroscopy data revealed that the R555W mutation causes a ‘flip’ of residue 555 from a surface exposed position in the wild-type to a buried position in the mutant.
The differences in stability and structure between FAS4 wild-type and mutants indicate that TGFBIp aggregation is, at least in part, caused by local changes in stability. The buried nature of the tryptophan residue is likely to explain the increased stability of the R555W mutant.
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