![]() ![]() One such mutation, C30R, breaks the C30-C41 disulfide bridge and results in OCA3. Most albinism-related mutations of Tyrp1 affect its stability or activity, and expansion of the interface between the EGF-like domain and the tyrosinase-like domain was linked to OCA Type 1B mutations in tyrosinase. It contains five total disulfide bridges, three of them located within the EGF-like fold. The core of the Cys-rich subdomain of Tyrp1 resembles the epidermal growth factor (EGF)-like domain due to its patterned disulfide bridges and short antiparallel ß-strands. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. ![]()
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