Molecular Dynamics Simulations Study On The Structure And Function Of Hyperthermophilic Protein

Thermophilic proteins have been one of the hotspots in the field of conputationalbiology and protein engineering, because of their thermal stability and uniquefunctions at higher growth temperatures. In this thesis, we applied moleculardynamics (MD) simulation to examine the structure and functions of Sso7d, Cren7,and Sto-Rnase HI. The content mainly includes the following three parts:1. MD simulation study on the thermal stability and conformations of DNA bythermophile protein Sso7dThermophile protein Sso7d in Sulfolobus solfataricu is a DNA-binding protein.Research shows that Sso7d is crucial for thermal stabilization and conformations ofDNA at higher growth temperatures. The details are still unclear.Focused on the two specific Sso7d-DNA complexes, we applied MD simulationsat four different temperatures (300,360,420, and480K) to examine the effect ofSso7d protein on the the thermal stability and conformations of DNA. The resultsshow that the protein Sso7d can help to stabilize the DNA molecules within a certaintemperature range. In addion to the thermal stability, the DNA molecules in the twocomplexes also undergo B-like to A-like form transitions with increased temperature,but the transitions only occur in part of the nucleotides.Based on the MD simulations at300and360K, we used the molecularmechanics/Poisson-Boltzmann surface area (MM-PBSA) approach to calculate thebinding free energies in the two Sso7d-DNA complexes. The results indicate (1) theextensive interactions between Sso7d protein and DNA phosphate backones, thereason why nonspecific DNA-binding protein Sso7d is proposed;(2) Lys7，Lys9，Lys22，Trp24，Val26，Met29, and Arg43are critical to the binding of Sso7d-DNA,which is consistent with the experimental results. This work can help to understandthe functions of Sso7d.2. MD simulation study on the interactions of Cren7-DNA (1) MD simulation study on the thermal stability and conformations of DNA bythermophile protein Cren7Cren7, a DNA-binding protein in crenarchaea, plays an important role in genomepackkaging and gene regulation. Research shows that Cren7is crucial for thermalstabilization and conformation of DNA at higher growth temperatures. But the detailsare still unclear.Focused on the two specific Cren7-DNA complexes, we applied MD simulationsat four temperatures (300,350,420, and480K) to examine the effect of Cren7protein on the the thermal stability and conformations of DNA. The results show thatthe protein Cren7can help to stabilize the DNA molecules within a certaintemperature range. In addion, the DNA molecules in the two complexes also undergoB-like to A-like form transitions with increased temperature, however, the transitionsonly occur in five nucleotides in3LWH and four nucleotides in3LWI.Based on the MD simulations at300and350K, we used the MM-PBSAapproach to calculate the binding free energies in the two Cren7-DNA complexes. Theresults indicate (1) the binding affinity for both the3LWH and3LWI shows anincreasing trend as temperature rise, which is consistent with the experimental resulatsof the two Sul7proteins;(2) the extensive interactions between Cren7and DNAphosphate backones are the reason nonspecific DNA-binding protein Cren7isproposed;(3) Lys24，Trp26，Leu28，Pro30，Lys31，Arg51，and Lys53make majorcontributions for Cren7-DNA binding. This work can provide important informationto understand the functions of Cren7.(2) MD simulation study on the effects of mutations on Cren7-DNA bindingThe experiments indicate the mutations of residues on the dsDNA-bindingsurface, such as W26, L28, and K53, which reduce binding affinity with the dsDNA.But the details are still unclear.We applied MD simulation and MM-PBSA calculation to explore the essence ofCren7-dsDNA interactions. Compared with the wild type Cren7, all the studiedmutants W26A, L28A, and K53A have obvious reduced binding free energies withdsDNA in the reduction of the polar and nonpolar interactions. In addition, thestructure and energy analysis indicate the mutations have a small effect to thebackbone structures but the decrease of the side chain interactions is responsible for the loss of the binding affinity.3. MD simulation study the effect of C-terminal tail on the structure and stabilityof hyperthermophile Sto-RNase HIThe C-terminus tail (G144-T149) of the hyperthermophile Sto-RNase HI playsan important role in this protein’s hyperstabilization, however, the effect of C-terminaltail deletion on the kinetics of thermal denaturation of Sto-RNase HI at elevatedtemperatures is still not fully understood.Focused on Sto-RNase HI and ΔC6Sto-RNase HI, molecular dynamics (MD)simulations at four different temperatures (300,375,475, and500K) were applied toinvestigate the unfolding process of Sto-RNase HI and ΔC6Sto-RNase HI. Theresults indicate that the deletion of the C-terminal tail in Sto-RNase HI changesubstantially the structure and overall fluctuations；the rate of unfolding is faster inΔC6Sto-RNase HI compared to Sto-RNase HI and the sequence of unfolding eventsis different in these two proteins.