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Rajamani Lakshminarayanan, Shyam S. Chaurasia, Jodhbir S. Mehta, Roger W. Beuerman; Analysis of Mutations in TGFβIp Associated Lattice Corneal Dystrophy Using Synthetic Model Peptides. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1101.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study is to understand the relationship between secondary structure, stability, and rate of aggregation in the transforming growth factor beta-induced protein (TGFβIp) associated lattice corneal dystrophy mutants.
We have synthesized a series of 23 residue peptides that describe β8 and β9 (611-633) of the 4th FAS1 domain of TGFβIp and 11 mutant peptides. Size exclusion chromatography (SEC) was used to assess the aggregation status of the model peptides. Secondary structure and conformational stability were assessed by far UV-circular dichroism (CD) spectropolarimetry. Fluorescence spectroscopy was used to assess the kinetics of aggregation.
Molecular dynamic (MD) simulation studies showed that the peptide formed anti-parallel β-sheet conformation by the same residues that are found in the crystal structure of 4th FAS1 domain. SEC showed no apparent changes in the aggregation status of all the peptides. CD studies showed that the wild type peptide exhibited a random coil structure in equilibrium with a β-sheet structure, confirming the MD simulations. Mutations induce varying effects depending upon the position of substitution and nature of the amino acid residues. Equilibrium guanidine hydrochloride denaturation assays demonstrated clear differences in the conformational stability of the peptides. All the mutant peptides, except G623R, showed marked decrease in conformational stability compared to the wild type peptide.
The results showed that all the lattice corneal dystrophy mutants (except G623R) in the β8 and β9 region of the 4th FAS1 domain lead to destabilization of the secondary structure, thereby, predisposing the protein to amyloidogenic conditions.
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