Effect Of The Middle Acidic Loop Region On Regulating The Structure And Function Of CYP38

Arabidopsis Cyclophilin38 (CYP38) is one of immunophilins located in the chloroplast thylakoid lumen. The first N-terminal 91 amino acid residues of the precursor protein is the signal peptide targeting the CYP38 protein into the thylakoid lumen. The structure of CYP38 mature protein contains two distinct domains:an N-terminal a-helical bundle domain (92-215 amino acids) and a C-terminal cyclophilin β-barrel domain (239-437 amino acids), connected by an acidic loop. The previous study showed that the full-length CYP38 and N-terminal domain can not interact with certain chloroplast proteins, including the AtCtpA protein, but the C-terminal domain alone can interact those proteins. The N-terminal a-helical domain is closely packed together with the C-terminal cyclophilin domain and establishes a strong intramolecular interaction, which further prevents the access of the cyclophilin domain to its targeting proteins. We believe that the middle acidic loop region plays a key role in mediating the interaction of the N-terminal domain and the C-terminal domain in the CYP38 protein.To test the physiological role of the acidic loop region in CYP38, we carried out studies in this thesis to is to observe whether CYP38 can interact with the chloroplast protein AtCtpA after several conserved acidic and basic amino acid residues in the loop region were altered to other neutral amino acid residues.First of all, CYP38 mature peptide sequence was amplified by polymerase chain reaction; then the DNA fragment was integrated into the prokaryotic expression vector pGEX4T.3 to construct the pGEX4T.3-CYP38 vector. Using the site direct mutagenesis methed, a number of mutations were introduced to the CYP38 coding sequences with pGEX4T.3-CYP38 recombinant plasmid as the template. Then I induced the corresponding fusion protein with GST tag product expression in E.coli; and I extracted and further purified the corresponding wild type and all mutated CYP38 proteins. At the same time the prokaryotic expression vector pET28a-AtCtpA was constructed in vitro to express the His tagged AtCtpA protein, and then I extracted and purified the AtCtpA protein. In the protein pull-down assay, the His taggedAtCtpA protein was used as the bait to test interaction between the AtCtpA and mutant CYP38 proteins. The protein pull-down experiments showed that the full-length CYP38 protein was not able to interact with the AtCtpA protein, but after basic amino acids in the loop chain region,236R and 238K were changed to alanine, glutamic acid or glutamine, the CYP38 protein can interact with AtCtpA. The other polar amino acid residues, including 216D,219D, 224E,227E,228E, and 230R were changed to alanine, individually or in combination, the CYP38 still can not interact with AtCtpA.I also tested the interaction between the AtCtpA and all the mutated forms of CYP38 in a yeast two hybrid assay. In detail, I constructed the pGBKT7-CYP38 with point mutations individually and pGADT7-AtCtpA vectors. Then I co-transformed pGADT7-AtCtpA and each mutated form of pGBKT7-CYP38 into yeast cells. The growth condition on the medium minus tryptophan, leucine, histidine and adenosine of each pGBKT7 and pGADT7 vector combination was recorded to show the interaction of AtCtpA and the mutated form of CYP38. We found that the yeast two hybrid experiments is consistent with results from the Pull-down, CYP38 protein can interact with AtCtpA when the basic amino acid residues in the loop region, 236R and 238K, were changed to alanine, glutamic acid or glutamine.The Pull-down and yeast two hybrid experiments show that basic amino acid residues in the loop region play a very important role in conformation and function of the CYP38 protein. We think that the two basic amino acids,236 K and 238 R, can regulate the interaction berween the N-domain and the C-domain in CYP38. We believe that the two basic residues, 236 K and 238 R, may sense the pH of thylakoid lumen caused by changes of chloroplast energy status. After 236K and 238R were protonated, CYP38 protein changes the intramolecular interaction N-terminal and C-terminal, resulting in the conversion of CYP38 between the active and inactive states.We planed to tested physiological function of mutated forms of CYP38 in plants. At first, DNA fragments coding mutated forms of CYP38 was created on the full-length coding sequence of CYP38 precursor protein, including the N-terminal transit peptide sequence. The binary vector pRIl 01-AN was adopted in this study, and all DNA fragement coding for mutated forms of CYP38 was constructed into pRI101-AN. The resulting vectors, pRI101-AN-CYP38 with each mutation, were transfered into Agrobacterium tumefaciens strain GV3101, and then introduced into cyp38-1, cyp38-2 mutant plants using the flower dipping method. Due to the time shortage and my inexperience, I am not ble to accomplish this experiment up to date, but I think it is very nessary and my current work laid a good foundation for further observing the phenotype of transgenic lines.