The Analysis Of Intramolecular Chaperone Of Nattokinase

Nattokinase (NK) is classified as subtilisin, which is a large class of serine protease. Since this family of proteases was found much earlier, and the members of the family are very large, various aspects of this family protease were researched. Some researchers considered the serine protease family as a model to study the catalytic mechanism of the enzyme. Other researchers put the eyes on the substrate specificity of the enzyme. These studies make us a good understanding of the characteristics and mechanism of the enzyme. With the development of structural biology, this family of proteases as a typical representative of the enzyme, many members of the structure has been successive resolved. We could analyze the serine protease family from the structure.Generally it is believed that the primary structure of the protein contains all of the information for the higher structure and biological function. This opinion also been verified by experimental results. However, due to the very complex protein folding, the study found that only part of the protein of all high-level structural information is contained in the amino acid sequence. Only rely on the primary structure, a lot of protein cannot has the high-level structure. They require the participation and assistance of molecular chaperones or intramolecular chaperone.These needs intramolecular chaperone protein, generally by the propeptide to act as this role. They interact with the mature peptide to form interaction interface, and perform the functions that facilitate folding. After performing function, propeptide is always hydrolyzed. Although they do not exist in the mature protein, the role of intramolecular chaperone make the change exists in the mature peptide. That is called protein memory. This structural variation in turn, can change the biological functions of proteins having different spatial structures. The same primary sequence of the protein is able to bind different spatial structures. Many proteins belonging to the subtilisin class have this phenomenon. Some research has been done, but these experiments are basically based on the process of in vitro protein refolding. After many experiment, we explore a method to express NK without the in vitro refolding. The mature NK can be obtained directly. Therefore, we use this method to study NK propeptide in vivo. We found the propeptide still perform intromolecular chaperone function. And from a series of experiments, we want to determine the most important amino acid during the precursor folding. Also, we try to reveal the molecular chaperone function mechanism, and to achieve the purpose of protein engineering by changing the Intramolecular chaperone.The alignment of the propeptide sequences of several different subtilisins revealed that three regions displayed significant sequence conservation and bind to four β-strands. non-conserved segments form two α-helices. First, we selected three amino acid residues in the propeptide by different homology. Form the site-directed mutagenesis and analysis of their structure and catalytic efficiency, it is revealed that the level of residue importance is consistent with the different levels of conservation of the residue. Secondly, we selected two amino acid residues in mature peptide to perform site-directed mutagenesis, and analyzed the structure information and enzyme kinetics. At the same time, we used bioinformatics as supporting research. After analysis, we found that in protein folding process, if amino acids in the mature peptide have interaction with the propeptide, such as the formation of hydrogen bonds, then the group of these amino acid changes can also affect the folding process, and will change the structure and function of mature protein. This reveals that the NK propeptide perform Intramolecular chaperone function by interacting with mature peptide. The point mutation experiments on the propeptide of NK show that protein folding in vivo is along with protein memory. This phenomenon exists not only in prokaryotes also in eukaryotes. This would inspire some understanding of the disease. In addition, we experimentally informed that it is different in structure and function between protein folding in vivo. The research of NK folding in vivo laid the experimental foundation for the subtilisin studies.For the study of intramolecular chaperone mechanism of NK, we performed fragment deletion and found that the conserved regions on the propeptide is essential. Conserved regions of propeptide guide the protein folding through hydrogen bonding interactions with some amino acids in the mature peptide. Through the study of the most conserved five amino acids in the propeptide, we determined four amino acids, Tyr10, Gly13, Gly34, and Gly35, play an essential role. Single point mutations of them can directly influence the the NK structure and activity. Multi-point mutatations can even completely block the mature peptide folding. Because of the high homology between the different family members, although NK was analysed as a model, it is likely that similar mechanisms exist for all subtilisins.After experiments and analysis, we have found some critical amino acids in the NK propeptide. Mutations of these amino acids can change the structure and function of NK through intramolecular chaperone mechanism. From the study of these amino acids, we can directly change NK to become with higher enzyme activity, greater stability, and wider applicability, to improve the application value of NK. At the same time, this research strategies and methods can be generalized to some other protease to increase their application utility.