Functional Analysis Of A Parvulin-type Peptidyl-prolyl Isomerases From Gossypium Hirsutum

Correct protein folding is very important for its biological function. In vivo, proteinfolding can be assisted by molecular chaperones and by foldases. Foldases include proteindisulfide isomerase (PDI) and peptidyl prolyl isomerases (PPI), which catalyze the cis-transisomerisation of peptidyl-prolyl bonds. The studies of PPI mainly focused on in mammals andin prokaryotes, while the research in plants is almost blank. The growth of plant is greatlyaffected by abiotic stresses. Proteins may lose their biological function because of unfoldingin these stresses, when chaperones and foldases can play very important role. Salt stress is abottleneck factor for plant growth. Many genes can be upregulated or downregulated undersalt stress, and the research of them may clarify the molecular mechanism of salt-tolerance. Acotton gene AY972810which is upregulated under salt stress was studied in this research, themain results are as follows:(1) Sequence homology analysis showed that the protein encoded by AY972810belongsto parvulin family. Trough BLAST in NCBI, we found that the C-terminal of this protein(residues48-140) have PPI domain. Amino acid sequence homology analysis bywww.expasy.org/tools indicated that the C-terminal (residues41-140) have the PPIconservative regions. The results of these two databases indicated that this protein may be amember of the parvulin family, so we designated it as GhPPI.(2) Phylogenetic analysis showed that this protein have very close evolutionary distancewith AtPin2and LjPar2. In order to determine the position of this protein in parvulin family,we mapped a phylogenetic tree using MEGA4.1software for homologous protein in19organisms. The result shows that this protein exibits91%identity to LjPar2and80%toAtPin2, rice and corn PPI. And it also has55%homology with hPar14and hPar17. Thesimilarity indicated that this protein is well conserved. In addition to C-terminal PPI domain,this protein also have N-terminal conserved regions through homology alignment, and it wasidentical with hPar14and hPar17. (3) GhPPI exhibit PPI activitie in vitro. This gene was cloned into the pET30a vector andthe fusion protein was overexpressed in E. coli BL21(DE3) cells. This protein was purifiedby His-tag affinity chromatography. The purified protein was used to assay PPI activity withthe substrate.The results showed that it has PPI activity which can catalyze the cis-transisomerisation of peptidyl-prolyl bonds., and the catalytic reaction rate depends on theconcentration of GhPPI. It is the first plant parvulin which is related to salt stress according toour NCBI database.(4) Speculating of the essential amino acid residues by homology modeling andmolecular docking.Three-dimensional structure models were built according to homologymodeling. Through structure comparison, we found seven active site residues which havesimilar positions to the other parvulin structures. We mimic the docking of substrate intoGhPPI using AutoDock4.0. And we find out eleven amino acid residues involving in substratebinding, which include the seven residues Speculated by homology modeling.(5) Identification of essential amino acid residues of GhPPI by chemical modificationand site-directed mutagenesis. Trough chemical modification, we determine the possibleessential amino acid residues are Cys, His, Met and Lys. We done site-directed mutagenesiswith possible active site residues. The results showed that the activity of seven mutants wasreduced. Combined with the results of chemical modification, molecular modeling andmolecular docking, we think that H48, H131, H134, F111, C90, M107and T130are theessential amino acid of GhPPI.(6) GhPPI exhibit chaperone activity in vitro. Using the aggregation of creatine kinase(CK) as a model system, we have shown that the GhPPI exhibits chaperone activity, beingable to suppress CK aggregation in vitro. And compared with the wild type GhPPI, thecapacity of suppressing CK aggregation of C90A and M107A decreased, H134A lost of thecapacity of suppressing CK aggregation completely. And the C90, M107and H134areessential amino acid for chaperone activity.(7) GhPPI is upregulated under salt stress, and our experiment proves it has isomeraseand molecular chaperone activity, suggesting that GhPPI may function as a molecularchaperone and folding enzyme in plant stress.