STABILITY STUDY OF DNA POLYMERASE I ITB-1 BASED ON MOLECULAR DYMAMIC AND SPECTROFLUOROMETRY APPROACHES

• Main Author: HELWATI (NIM: 30504009), HIRA
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/12215
• Institution: Institut Teknologi Bandung

DNA Polymerase I ITB-1 is a member of DNA polymerase I family. The enzyme was isolated from thermophilic bacteria from Cimanggu Crater, West Java. Based on 16S rRNA analysis, the bacteria has highest similarity to Geobacillus thermoleovorans. DNA Pol I ITB-1 gene has been cloned and expressed in Escherichia coli, consists of 2682 nucleotides that encodes 876 amino acid residues. Amino acid sequence homology of DNA Pol I ITB-1 indicated that the enzyme has highest similarity to all known DNA Pol I, with the highest homology to DNA Pol I from Bacillus caldoliticus. Homological analysis revealed that the protein contained all of the motive for 5' -> 3' exonuclease, 3' -> 5' exonuclease and 5' -> 3' polymerase domains. Crude extracts of the expressed protein showed optimum activity at 65oC (338K) and pH 7.4. Structure and function studies of the DNA Pol I ITB-1 have been carried out either through biochemical or the computational analysis using molecular dynamics simulation. However, there is no information reported concerning domains DNA Pol I ITB-1 when expressed and analyzed separately. In this research, the stability of DNA Pol I ITB-1 structure, particularly the individual structure of DNA Pol I ITB-1 domains that performed separately were carried out by molecular dynamics simulation and experimental approaches.<p>Three-dimensional structure of the Klenow-like DNA Pol I ITB-1 obtained from the Swiss-model (http://www.expasy.org/Swiss-Model) server has high similarity with the Bacillus Fragment (BF) structure, especially with B. stearothermophilus. Klenow-like DNA Pol I ITB-1 consists exonuclease 3' -> 5' at the N –terminal and polymerase 5' -> 3' at the C-terminal domains, meanwhile the polymerase consists thumb, fingers and palm subdomains. Theoretically, both domains (exonuclease 3' -> 5' and polymerase 5' -> 3' were separated based on amino acid sequences, however for experimental purposes, the deletion mutants were constructed by removing the certain domain on DNA Pol I ITB-1.<p>Molecular dynamics simulation of the Klenow-like DNA Pol I ITB-1, 3' -> 5' exonuclease and polymerase 5' -> 3' domains at 300K, suggested that all of structural models were stable. Analysis of Ca-RMSD (root meat square deviation) for all protein models did not show significant change in the structure of the proteins. The result was supported by secondary structure and solvent accessible surface area (SASA) analysis at 300K showed no significant change during the simulation (10ns). Further analysis on flexibility of individual amino acid residues in the each of the proteins was calculated by root mean square fluctuation (RMSF). The RMSF analysis of both separated domains (3' -> 5' exonuclease and polymerase 5' -> 3') showed similar fluctuation compared to that the Klenow-like DNA Pol I ITB-1 structure. These results suggested that both domains were stable and may have an independent folding at 300K. MD simulation at higher temperatures were performed in order to investigate thermal stability of both domains separately and compared to that Klenow-like DNA Pol I ITB-1. Based on RMSD analysis of simulation at higher temperatures suggests that polymerase domain was the most unstable one. It was indicated by RMSD value of polymerase domain reached higher value faster than that of two other protein models. RMSD analysis at 360K suggested that the structure of 3' -> 5' exonuclease domain took longer time to unfold than polymerase domain. The MD simulation suggested that the individual domains of DNA Pol I ITB-1 particularly, 3' -> 5' exonuclease and polymerase 5' -> 3' domains were stable at room temperature, however, these domains depend on each other to maintain the stability of the Klenow-like DNA Pol I ITB-1.<p>For the experimental purposes, deletion mutants of DNA Pol I ITB-1 were constructed by removing part of the gene resulted DEcoRI and DXhoI mutants. The DEcoRI mutant was losing part of polymerase domain, meanwhile DXhoI mutant donot have most 3' -> 5' exonuclease domain. The genes have been expressed in E.coli through pET30a (+) expression vector. The expressed proteins were purified using Ni-NTA matrix. Conformation stability analysis of the wild type and DEcoRI mutant were performed by spectrofluorometry analysis based on intrinsic fluorescence properties of thryptophan residues. The whole DNA Pol I ITB-1 have five Trp residues that ditributed in 3' -> 5' exonuclease and polymerase 5' -> 3 domains. The flouresence intensity of the proteins in various pH conditions showed that the wild type protein has highest intensity at pH 7.5, meanwhile the mutant showed at pH 7.0, this is suggested that the wild type and the mutant showed the highest compactness at the respective pH. The compactness of both of proteins decrease dramatically when the pH change to be more acid or base. Fluorescence quenching analyses suggested that the DEcoRI showed more compact structure compared to that the wild type protein. It is indicated by Sterm-Volmer constant value of the deletion mutant in all pH lower than that the wild type, however the DEcoRI was more sensitive at pH change compare to the Wild type.<p>Stability studies of the of DNA Pol I ITB-1 structure by theoretical and experimental approach suggest that 3' -> 5' exonuclease and 5' -> 3' polymerase domains have important roles for stability of a whole protein, particularly the Klenow-like fragment. The MD simulation analysis at higher temperature showed that the Klenow-like DNA Pol I ITB-1 structure was more stable than individual 3' -> 5' exonuclease and 5' -> 3' polymerase domains. On the other hand based on the experimental results, the structure of exonuclease 3' -> 5' (presented by EcoRI deletion mutant) is more sensitive toward environmental pH change compare to those of DNA Pol I ITB-1 wild type. These results sugested that both domains were have importance role in maintance the whole structure of DNA Pol I ITB-1.