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K. Runager, J. Underhaug, R. V. Basaiawmoit, Z. Valnickova, I. B. Thøgersen, H. Karring, G. K. Klintworth, D. E. Otzen, N. C. Nielsen, J. J. Enghild; Phenotypically Distinct Corneal Dystrophies Link to Differences in the Thermodynamic Stability of the Fourth FAS1 Domain of Transforming Growth Factor Beta Induced Protein (TGFBIp). Invest. Ophthalmol. Vis. Sci. 2010;51(13):4294.
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Point mutations in the TGFBI gene lead to several phenotypically distinct corneal dystrophies. In the present study we investigated the thermodynamic stabilities of recombinant wild-type (WT) and mutant TGFBIp.
Recombinant WT and mutant TGFBIp were expressed in mammalian cells while WT and mutants of the fourth FAS1 domain (FAS1-4) were expressed in a bacterial expression system. Proteins were then purified under non-denaturing conditions and the thermodynamic stabilities were assessed by transverse urea-gradient (TUG) gel electrophoresis as well as thermal and chemical denaturation monitored by far-UV circular dichroism (CD) spectroscopy. The solution structures of WT and mutant FAS1-4 were solved using nuclear magnetic resonance (NMR) spectroscopy.
Biophysical analyses of TGFBIp with naturally occurring mutations in the FAS1-4 domain displayed clear differences in their thermodynamic stabilities. Specifically the A546T mutant, giving rise to amyloid depositions in vivo, was less stable than WT while the R555W mutant, which is accompanied by crystalloid protein depositions was significantly more stable than WT TGFBIp. The R555Q mutant, which forms small irregular aggregates (‘curly fibers’) in vivo, displayed an intermediate stability. These differences were found to result from local stability changes in the FAS1-4 domain as was shown by analyzing this domain separately. By solving the solution structures of WT and R555W FAS1-4 we further showed that the increased stability of R555W is due to stabilization of the hydrophobic core of FAS1-4 by the mutated amino acid residue.
The ability of mutant TGFBIp to form a diverse array of phenotypically distinct protein deposits is intriguing. In the present study we show that differences in thermodynamic stability are likely to account for the diverse types of protein depositions formed by mutants of the FAS1-4 domain of TGFBIp. Accordingly, three phenotypically distinct TGFBIp mutants displayed three different thermodynamic stabilities, which all differed from WT TGFBIp.
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