| Collagen is the most abundant protein in mammals and an important component of extracellular matrix.Collagen is a linear structural protein,three left-handed helixes?chains intertwine to form a right-handed supercoiled structure.Collagen can be divided into homotrimer and heterotrimer according to whether the three chains are the same.In addition to being structural protein in the body,collagen is closely related to many diseases such as osteogenesis imperfecta,arthritis and immune diseases.Therefore,imitating the weak interaction between natural collagen sequences,designing new collagen sequences and establishing research platforms are of great significance to the study of collagen diseases and the design of collagen materials.In this study,the heterotrimer abc,which was designed by our group before,was used as the host to study the specific design and folding mechanism in collagen backbone and side-chain.The relationship between the stability of collagen triple helix under different forces was proposed to provide a theoretical basis for the design of new collagen heterotrimer.The specific content is divided into the following five parts:(1)New collage-like peptides sequences were designed based on abc heterotrimer.34 new peptides were designed to study the stability of natural amino acids in Y and X position,16 new peptides containing salt bridge were designed to study the effect on the thermal stability,15collage-like peptides and 6 groups of new sequence were designed to learn the influence of cation-?interaction and hydrogen bonding interaction on the thermal stability respectively.(2)The results of circular dichroism analysis showed that Ile,Val and Ala exhibited high stability at Y and X sites(T_m value was greater than 30?).Aromatic amino acids like Phe,Tyr and Trp had obvious destructive effects on the stability of trimer(average T_m value reduced by6?).Due to the large number of charged residues in the sequence of heterotrimer abc,the average thermal stability reduced by 5.8?among these mutations(b10R?c9K and c9D).The amino acids with high frequency and stability of natural collagen were Ala,Gln and Thr in Y position,Ala,Leu and Glu in X position.These amino acids can be used to design collagen-like peptide with high stability.(3)The data of thermal stability showed that among 16 pairs of salt bridges,Four types of axial salt bridge interaction had electrostatic attraction,the magnitude of which was KD>KE=EK>ER,the axial KD and KE salt bridges also had a high frequency of occurrence in natural collagen.The predicted T_m values of bacteria collagens calculated by the contribution values of salt bridge interaction were 25?and 23.6?,respectively.The T_m values of collagens expressed in E.coli.were measured by the circular dichroism,the results were 25.8?and 24.7?respectively.The stability of type I collagen was predicted according to the experimental value of the salt bridge interaction.Heterotrimer?2?1?1 and homotrimer?1?1?1 had the most stable structure.(4)Different combinations among Phe,Tyr,Trp,Arg and Lys were studied in heterotrimer abc.Five axial forces FK/YK/WK/RF/RW and lateral force RF had a stable interaction on collagen structure.Axial RW had the highest thermal stability(5.5?).The highest occurrence rate among 6 pairs of forces in natural collagen was axial RF(1.71).(5)Research the hydrogen bonding forces of Lys and Asn side chains in homotrimer and heterotrimer.The contribution values of hydrogen bonding forces to the stability of homotrimer and heterotrimer were 2.8?and 6.5?,respectively.Molecular dynamics simulation results showed that side chain hydrogen bonding existed in both systems with probabilities of 0.688and 0.357,respectively. |
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