Application Of Molecular Evolution And Docking In Drug Screening

The studies on the evolution of structures have been around for a long time, but only recently have significant progress been made.Carbonic anhydrase (CA, EC 4.2.1.1) are zinc-containing enzymes catalyzing the reversible hydration of CO2. They are ubiquitous in Eukarya, Archaea and Bacteria domains. The enzyme is important to many physiological processes such as respiration, CO2 transport and photosynthesis. CAs had been used as the important target proteins for drug designs.Protein structure is more highly conserved than sequence and it can give more detailed evolutionary information, the comparative analysis of structures allows the investigation of evolutionary events. So, this dissertation studied the molecular evolution ofα-,β-, andγ-class carbonic anhydrases based on their domain structures.To obtain a reliable analysis, we defined a subset that contains all specificities and organisms as the nonredundant structure set using QR factorization based on the multiple structural alignment of the known crystallographic structures ofα-,β-, andγ-CAs with QH and Qres as the structural homology measure. Then, we applied unweighted pair group method with arithmetic averages (UPGMA) to reconstruct structural phylogeny with 1-QH as the distance. Further analysis of the catalytic domain requires importing the sequences as well. This dissertation did so by performing a BLAST search of a carbonic anhydrase domain structure from the nonredundant structure set agaist the Swiss-Prot database one at a time. The over-represented sequences were eliminated. Using ClustalW to align the sequences and use MultiSeq to keep only the portions of the sequences that aligned to the catalytic domain in the structures. The sequence QR factorization was used to select the nonredundant sequence set. Finally, the structural alignment of the nonredundant structure set was used to guide the sequence alignment of the noredundant sequence set. The phylogenetic trees were reconstructed using neighbor-joining method with poisson distances as the metic.Then, this dissertation discussed the effect of cell culture on the Taxus chinensis cells 18S rRNA gene based on the phylogenetic analysis and molecular clock hypothsis. At last, we validated the functional group from evolution of structures via virtual screening based on molecular docking.The results suggest as follow:1.α-CAs are from a superfamily with highly structural homology, with QH > 0.4 and Sc > 5.5. Only the core structure of active site ofβ-CAs is highly conserved, and theγ-CA belongs to a very diverse superfamily of proteins that share the left-handedβ-helix (LβH).2. The protein moleculars with different sequences either can be in possession of the similar structures with the same function, or can bear different functions with some semblable structures. It is worth to note that the key residues in sequence have more important influence on protein function than structure. The relationships among sequence, structure and function provide theoretical principle for applied research of CA, such as the alteration of CA activity, and the drug design etc.3. The substitution of resides in active site can change the activity of CA, and the specificity of active site can help for the specific-drug design. The conserved and specific regions of active site give highlight to the applied research of CAs, such as the alteration of CA activity, and the drug design etc.4. The domain ofγ-CA underwent a process ofα-helical content from amino- terminal end to carboxyl-terminal end of the left-handedβ-helix; the capacity ofγ-CA to bind Zn occurred early in evolution and only later included the ability to catalyze the reversible hydration of CO2 efficiently for the occurrence of two loops involving Glu 62 and Glu 84 respectively and a long helix at the carboxyl-terminal end of the LβH.5. The structural similarities are sufficient to allow recognition of CsoSCA as a member of theβ-class of carbonic anhydrase. More than that, the structural analysis strongly surpports for the CsoSCA should qualifiy as a distinct subclass ofβ-CAs.6. Cell cultures make the evolution of Taxus chinensis cells 18S rRNA gene faster, and the divergence time between HG-1 and T. chinensis is about 7Ma.