| Summary: | 碩士 === 國立成功大學 === 生物科技研究所 === 90 === Studies of the secondary structures of Human positive cofactor 4 (PC4)
In the process of protein synthesis in cells, mRNA must be used as template to translate intact proteins in correct sequence. Therefore, synthesis of mRNA is an important process in the regulation of protein synthesis in organisms. In the process of transcription, synthesis of mRNAs need some transcriptional factors participate in the reaction. The major role of transcriptional factors is to help RNA polymerase bind to the promoter of DNA template in the beginning of transcription. Otherwise, transcriptional cofactors also participate in the reaction by binding on the upstream of promoter in an activated DNA template, and then regulate the binding reaction between RNA polymerase and the promoter of DNA template.
Positive cofactor 4 (PC4) is a transcriptional cofactor composed of 127 amino acids. PC4 can make a synergistic effect with TBP-associated factors (TAFs) and regulate the interaction between transcription-activating factors and transcriptional factors。Unphosphorylated PC4 is the functional form in positive-regulated transcription, and phosphorylated PC4 can not regulate transcription (Ge et al. 1994).
In this study, PC4 gene was cloned into pET-21a vector and expressed in E coli BL21 strain. The secondary structures of PC4 were monitored by infrared (IR) and circular dichroism (CD) spectroscopy, and furthermore to confer the binding mechanism affected by the characteristics of secondary structures of PC4。The results have shown that the predominant secondary structure of PC4 is a-helix, and there are still little parts of PC4 in b-sheet structure. Based on the results of this study, we can further confer the factors that three dimensional structure of PC4 mediated by its secondary structures.
Studies of the secondary structures of a1-antitrypsin (AAT) C-5 peptides
Fragments from various proteolytically degraded precursor proteins can form b-amyloid fibrils. The phenomenon of peptides’ aggregation in the form of b-amyloid fibrils may be related to their secondary structures. Therefore, we synthesized many peptides to study the characteristics of their secondary structures.
a1-antitrypsin (AAT), the predominant serine proteinase inhibitor (serpin) in human serum, is an inhibitor of serine proteases in general but its most important target enzyme is neutrophil elastase. The hydrophobic C-5 peptide in hydrolyzed C-terminal peptide compos of 5 amino acids (FVFLM), aggregates and forms deposit similar with amyloid deposit. Therefore, we have synthesized 20 wild-type and constant C-5 peptides which the amino acid were replaced with a particular amino acid at the third position, in order to study the effects of different amino acid on the formation of secondary structures of the peptides. The information can be used further for structure-based drug design.
The results of IR and CD spectroscopy have shown the secondary structures of these twenty peptides are different because of the characteristics of the replaced amino acid. Most peptides aggregated and formed a-helical structure, b-sheet and random coil at different ratios. The main secondary structure is b-sheet, especially for those peptides having nonpolar, aliphatic amino acid. From the results obtained, we can understand more about the factors of different characteristics of amino acid determining the formation of secondary structure.
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