Study On The Structural Stability Of Proteins By High Temperature And Site Directed Mutagenesis

Protein is one of the most important components of organisms and it almost participates in all the lifeprocess and cell activity. The protein plays important role in many fields, such as signal transduction, cellmetabolism, cell adhesion, individual growth and development and protein-protein interactions, etc. Mostproteins have a tendency towards disassemblize and reassemblize, which are affected by various conditions,and the dynamic property is a prerequisite for the protein to function properly. Therefore, the structuralstability of the protein is crucial to the dynamic function of its. In this thesis, Transmissible SpongiformEncephalopathies (TSE), Insulin-like Growth Factors (IGFs) and IGF-binding proteins (IGFBPs) wereselected. Molecular dynamics (MD) and steered molecular dynamics (SMD) simulations are performed toinvestigate the relationship between the interaction of the intermolecular and intermolecular and structuralstability of the protein, as well as study on the higher structural stability of protein by high temperature andsite directed mutagenesis. It is might provide more insight into the interaction mechanism of protein-proteinsystems, and helpful for the diabetes based drug design and the detection of the key interaction site.This paper is mainly composed of the following five parts:Chapter1: The protein and protein-protein interaction were briefly introduced, as well as summarizes themethods that the structural stability of protein and the mainly contents.Chapter2: The methods of MD combined with SMD simulations were introduced.Chapter3: Investigation of MD simulation and alanine mutations affecting insulin-like growth factor (IGF)I binding to IGF binding proteins were studied in detail. It was found that the binding sites in dynamicsstructure were changed to compare with the static X-ray structure of IGF-I and IGFBPs, as well as MD simulation was helpful to understand the interaction mechanism of IGF-I and IGFBPs at the atomic level.The six key residues in IGF-I were screened and compared with the binding interactions byAlanine-scanning mutagenesis. The positive and negative mutation effects showed that the biochemical orbiological properties of Ala substituted IGF-I mutants could not be used in a straightforward way to dissectthe direct involvement in binding of individual residue since conformational and structural changes might beinvolved.Chapter4: Single-point and continuous multiple mutations were combined to study the interaction and thedistribution of key sites between IGF-I and IGFBP4, as well as investigation of the binding interaction bypolarity site directed mutagenesis. It was found that the reduction of the helix content was not inevitable forthe mutants, and mutants could not be used in a straightforward way to dissect the direct involvement inbinding of individual residue since conformational and structural changes might be involved. Therefore, itwas not appropriate to use the site dircted mutagenesis to study the biological of individual residue. Theontinuous multiple mutations results also showed that no obvious cumulative effect appeared with theincrease of the mutant sites. And it could be regulated by careful selection of the substituted amino acid.Chapter5: Molecular dynamics simulation of temperature induced unfolding of animal prion protein. Byperforming at different temperatures, the unfolding mechanisms of two prion proteins were studied tounderstand the molecular basis of disease-related structural transition. It was found that the unfolding orderof the secondary structure at different temperature was similar. And the β-sheet was rather active with theelongation and disruption during the simulation. And the unfolding of β-sheet occurred more readily at theelevated temperature. The unfolding dynamics of α-helix was shown to begin from ends to the middle, andthen the whole helix was unfolded. And the conversion of α-helix to π-helix was found to be common at the elevated temperature. The content of helix was gradually decreased with the elevation of temperature,which showed that α-helix response to the temperature of proteins sensitive at elevated temperature.