The Effect Of The Incidence Of Cis-Peptide Bonds In Flexible Regions On The Thermal Stability Of Proteins

The thermal stability of proteins is not only related to the interaction between molecules such as hydrogen bonds,disulfide bonds,ionic bonds,hydrophobic interaction,but also the surrounding environment and the folding and unfolding process of proteins.One of the important factors affecting the folding and unfolding process of proteins is the structure of the rigid and flexible regions of proteins.So it may be hindered by enhancing the rigidity of the flexible regions of proteins.Thereby it can increase the tolerance of proteins to temperature.The types of peptide bonds that make up proteins can be classified into cis-peptide bonds and trans-peptide bonds.The cis-peptide bonds have a natural bending structure,which may maintain the stability of the structure when the internal stress of proteins weakened or disappeared.It has been found that natural proteins exhibit trans-conformation in most cases.Studies also have found that physicochemical,biological,genetic and other factors may lead to amino acid substitution during protein biosynthesis,which may result in the cis-trans isomerization of proteins.The cis-trans isomerization of proteins is not a static process,but the dynamic process is difficult to observe.At present,there is no scientific method to determine the effect of cis-trans isomerization on the thermal stability of proteins.Combined with the above theory,it can be inferred that introducing cis-peptide bonds into the flexible region of proteins may improve the thermal stability of proteins,which is still lack of evidence.Based on the study of the cis-trans isomerization process of proteins,our group proposed the concept of incidence of cis-peptide bonds(ICPB),and used the cis-peptide bond incidence to describe the results of the dynamic process of cis-trans isomerization.Studies have shown that although ICPB was usually dependent on environmental factors such as molecular chaperone,post-translational modifications and so on,which made it different in prokaryotic and eukaryotic cells.In the early stage of this project,a data set of human protein structure without redundant data was established by using the Protein Database(PDB).Then,the bioinformatics method was used for statistical analysis.It was found that the incidence of cis-peptide bond(ICPB)was closely related to proline.Comparing the incidence of cis-peptide bonds of different kinds of X-Pro,it was found that ICPB was higher when X was an aromatic amino acid,which was lower when X was an aliphatic amino acid,methylated orubiquitinated modified amino acid(methionine,leucine,histidine,etc.),that is,cis-peptide bond was not easy to occur.Based on the previous theoretical speculation and the previous research conclusions of our group,we speculated that the thermal stability of protein could be improved by mutating the former amino acid into a higher amino acid of ICPB after introducing proline into the flexible region of protein.In order to prove the hypothesis,this study mainly carried out the following experiments:(1)A same protein(enzyme)dataset derived from thermophilic and mesophilic organisms was constructed by Swiss-Prot database.Then,statistical analysis by bioinformatics method was carried out to obtain the peptide bond species from different temperatures under natural conditions.(2)Using Luciferase from North American firefly as the experimental research object,the mutation site H489 P was introduced in flexible region.Combining with the previous research conclusion on ICPB,other amino acid mutations(E488W-H489 P,E488Y-H489 P,E488M-H489 P,E488H-H489P)were introduced in the first position of amino acid 489 to construct proteins containing different kinds of peptide bonds.(3)The wild-type and mutant luciferase were assayed for kinetic constants and half-life of protease at different temperatures to identify the changes in the thermostability of protease,observed the relationship between the thermal stability of proteins and the incidence of cis-peptide bonds.Based on the above research,the main results of this paper were as follows:(1)A data set of DNA polymerase I(EC 2.7.7.7)was constructed in this study,which counted from 6kinds of thermophiles and from 31 kinds of mesophiles.And A data set of Alpha-amylase(EC3.2.1.1)was also analysed in this study,which counted from 4 kinds of thermophiles and from20 kinds of mesophiles.(2)After extracting the amino acid characteristics form EC 2.7.7.7,it was found that thermophilic proteins contained a larger proportion of A、E、G、H、L、P、R、V and W than mesophilic proteins.Among them,H、G、L、P and R showed significant differences,which was indicated that the thermal stability of DNA polymerase I might be closely related to many kinds of amino acids.After extracting the characteristics of proline-related peptide bonds,it was found that thermophilic proteins contained a larger proportion of A/D/E/F/H/K/L/M/P/R/T/V/W-P than mesophilic proteins,of which significant differences were found in EP、KP、AP、DP、FP、HP and PP,and QP showed a significant decrease.The results are consistent with the rule of ICPB.Therefore,we think ICPB may be the main factors of DNA polymeraseⅠthermal stability.(3)After extracting the amino acid characteristics form EC 3.2.1.1,it was found that thermophilic proteins contained a larger proportion of D、E、F、H、I、K、L、Q、R、W and Y than mesophilic proteins.Among them,F、H and W showed significant difference,which was indicated that the thermal stability of Alpha-amylase might be closely related to F、H and W.After extracting the characteristics of proline-related peptide bonds,it was found that thermophilic proteins contained a larger proportion of C/E/F/H/I/K/V/W/Y-P than mesophilic proteins,of which significant differences were found in EP、FP、HP and IP.However,SP and TP showed a significant decrease.Amongthem,the results of EP,TP and FP were basically consistent with ICPB,while the results of HP,IP and SP were not consistent with ICPB.We hypothesized that it might be because ICPB was not the most important factor affecting alpha-amylase.(4)The experimental results showed that introducing of the mutation site H489 P into the flexible region of luciferase of firefly in North America can significantly improve its thermal stability.The specific performance of the half-life is delayed.(5)The luciferase that mutated the 488 th amino acids into W/Y has a higher thermostability trend than the luciferase mutating into M/H at this position.The specific performance of the half-life is delayed,which was basically consistent with the law of the incidence of cis-peptide bonds.In summary,this study preliminarily demonstrated that introduction of peptide bond type with higher ICPB into flexible regions of proteins may increase the thermal stability of proteins,which provided a direct evidence for the influence of different ICPB peptide bond types on the thermal stability of proteins.The impact further provided new ideas for the modification of the thermal stability of enzymes.