Modifications on protein termini of bacillus thermocatenulatus lipase and their impacts on activity and stability
Yılmaz, Hazal (2014) Modifications on protein termini of bacillus thermocatenulatus lipase and their impacts on activity and stability. [Thesis]
Bacillus thermocatenulatus lipase (BTL2) is a thermostable enzyme with a known three dimensional structure (PDB ID: 2W22). The N- and C- termini of protein backbone in this structure seated very close to each other (<5 Å), unlike to other lipase structures having their termini located fairly apart from each other. For other proteins that possess circular backbone, the close proximity of the protein termini has been shown to contribute to thermal stability. From this perspective, the protein termini and particularly its impacts on lipase stability are investigated in this thesis. During these investigations, three BTL2 variants are used and explicitly these are N7G, N7Q and R5C-A6C-N5C-S386C-L387C-R388C. The closest contact of the backbone termini is a hydrogen bond formed by the side chain of 7th asparagine and the main chain of L387. In the first two mutations N7G and N7Q, the impact of this hydrogen bond is investigated, while in the third mutation three consecutive residues from the N-terminus (5-6-7) and from the C-terminus (386-387-388) are substituted with cysteines aiming to induce a disulfide bond in the third mutant. Along with the native BTL2, three mutants are obtained in high purity via application of various molecular biology and protein engineering routines including site-directed mutagenesis, ligation-independent cloning, heterologous protein expression and affinity purification methods. The native BTL2 and the mutants are subsequently characterized in enzyme activity assays to determine their thermoactivity, thermostability and substrate selectivity profiles. Furthermore the far-UV circular dichorism (CD) spectra are collected for all lipases to analyze their secondary structure and melting temperatures. The results indicated that all three mutations did not have any significant effects on thermal stability, thermoactivity and substrate selectivity of native BTL2 suggesting that the modification of the hydrogen bond at the lipase termini is not related to the integrity and thermal stability of the catalytic domain. Different from the hydrogen bond mutants, the third mutant showed significant decrease in the thermoactivity and thermal stability of the native BTL2. This particular finding suggested that the cysteine substitutions at the termini caused destabilization of the active site and overall structure of the lipase. Considering the possible implications of the modification of the protein backbone such as generation of protein analogues with optimal stabilities, this thesis aimed to analyze the impacts of the modifications of the termini on the lipase characteristics. Overall, it has been concluded that such modifications of the termini would be useful in generation of lipase variants with optimal features without affecting the core domains.
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