Studies On The Folding And Structure Of Arginine Kinase From Greasyback Shrimp(Metapenaeus Ensis)

Arginine kinase (AK)(ATP:L-arginine phosphotransferase EC2.7.3.3) is a member of the phosphagen kinase family and a homolog of creatine kinase (CK). It participates in cellular energy metabolism and catalyzes the reversible phosphorylation of arginine by MgATP to form phosphoarginine and MgADP in invertebrates.Studies on CK have shown that it exists in two forms, the reduced form (R-CK) and the oxidized form (O-CK), and O-CK contains an intrachain disulfide bond in each subunit. The generation of O-CK was a negative regulation of R-CK that O-CK can be rapidly ubiquitinated and degraded by ubiquitin-dependent pathway. AK contains five free cysteine thiols in the molecule. It is unclear yet whether AK has the reversibly oxidized form and O-AK has intramolecular disulfide bonds or not. In the present study, we have first confirmed that AK from Greasyback Shrimp (Metapenaeus ensis) and its mutant R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K). We found that the R330K mutant was more susceptible to oxidation than wild type AK (Wt-AK), and post-added DTT can reverse O-R330K to the reduced form, which suggested that the oxidation might be mediated by reversible disulfide bond formed between cysteines. To identify the cysteine residues responsible for the disulfide bond formation in O-AK or O-R330K, five site-directed mutants of Wt-AK (C23S, C127S, C139S, C201S, C271S), and five site-directed mutants of R330K (C23SR330K, C127SR330K, C139SR330K, C201SR330K, C271SR330K) were independently constructed and analyzed on reduced and non-reduced SDS-PAGE. It can be concluded that the intramolecular disulfide bond of O-AK or O-R330K was formed between Cys201and Cys271.The roles of some amino acid residues have been investigated on AK in terms of structural stability and catalytic functions. Multiple sequence alignments indicated that nine positively charged Arg residue (R124, R126, R129, R208, R229, R245, R280, R309, R330) are highly conserved in AKs and CKs. Accumulating evidence suggests that the five conserved arginines (R124, R126, R229, R280, and R309) are associated with the catalysis of phosphoryl transfer. One exception is R330, which is strictly conserved within phosphagen kinase family founded at present. It is not clear whether R330only plays a general role in the structural maintenance of AK. In order to investigate the role of R330in AK, it was replaced by lysine (R330K). Biochemical analysis revealed that decreased secondary structures, changed microenvironment of Trp residues and increased hydrophobic surface exposure of R330K were concomitant with the sharp decline of catalytic activity, and the results were further confirmed by structure modeling of AK and R330K. Our findings provided the insights into the structural and functional roles of R330residue of AK. It can be concluded that R330residue plays an important role in the structural stability and activity of AK.Aggregation of AK could be induced by heat treatment or occurred in the refolding process of AK denatured with GdnHCl. Molecular chaperone helps other protein avoid misfolding pathways that produce aggregated conformation. FK506binding protein12is a small single domain protein possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. There is controversy whether FKBP12also possess chaperone activity or not. In this study, wild-type human FKBP12(Wt-hFKBP12) protein was purified and analyzed with size exclusion chromatography (SEC), glutaraldehyde cross-linking, and cetyltrimethyl ammonium bromide electrophoresis (CTAB-PAGE). The results suggested that Wt-hFKBP12exists in forms of monomer, dimer and trimer. Subsequently, hFKBP12(His), recombinant protein with six histidine-tagged, was constructed and purified. The PPIase activities and thermal stability under65?of Wt-hFKBP12and hFKBP12(His) were basically the same. We investigated the aggregation of AK in the presence of hFKBP12. The results indicated that hFKBP12could not suppress heat-induced aggregation of AK or aggregation occurred in the refolding process of AK denatured with GdnHCl.Macromolecular crowding is an ubiquitous phenomenon in living cells that significantly affects the function of enzymes in vivo. However, this effect has not been paid much attention in the research of the refolding of AK. To study the effects of macromolecular crowding on refolding of recombinant AK, PEG2000, Dextran70and Ficol170were selected as macromolecular crowding agents. The results suggested that: three kinds of macromolecular crowding agents (100g/L) could accelerate the fast phasic reaction of the refolding of urea-denatured AK, while the final refolding yields of AK decreased.