Effect Of Interchain Salt Bridges On Thermal Stability Of Collagen

Collagen is the most abundant protein in higher organisms,with a characteristic three-strand helix structure.Most of the known types of collagen are homotrimers,i.e.,the collagen triple helix consists of three identical chains,while the heterotrimeric triple helices are more prevalent than homotrimeric ones.The main interactions maintaining the stability of the triple helix are the hydrogen bond,preorganization of amino acids,salt bridge,and Van der Waals’forces.The charged amino acids have high occurring frequencies in natural collagen,suggesting that the salt bridges may play an essential role in collagen folding.Also,there are complex salt bridges in natural collagen,with three or more charged residues interplay simultaneously,which increase the complexity of the system greatly.In this study,a collagen heterotrimer abc designed previously by the research group was selected as the host peptide to explore the molecular mechanism of interchain salt bridges of collagen stabilization.A discrete computational model with collagen scoring function was established,and guide the rational design of heterotrimer collagen-like peptides successfully.This research mainly contains several aspects as below:(1)In this study,an isolated complex salt bridge in host heterotrimer abc was selected for rational site-specific mutagenesis.Measuring circular dichroism(CD)spectra of 17 variants to determine their thermal stability.For the single salt bridges led by K,the axial KD’ and KE’were constantly strong,followed by the weaker lateral KE,while the lateral KD showed a marginal contribution.The R-led single salt bridges made modest contributions,ΔTm values of RE,RD’,and RE’ were similar and slightly higher than that of RD.The strength of the K-anchored complex salt bridges was stronger than that of the R-anchored ones,and the eight complex salt bridges showed consistent anti-cooperativity.(2)Construct molecular models for the seventeen variants and perform molecular dynamics simulations.The dynamic characteristics such as formation probability of sidechain electrostatic interactions,sidechain-backbone hydrogen bonds,and probability that the sidechain dihedral angle χ4 of the Y-site residue forming trans(t)conformation were analyzed.Multiple linear regression was conducted between the statistical probabilities and the thermal stability of collagen to analyze the molecular mechanism of the energetic contribution of different types of the salt bridge.The results showed that there was a strong correlation with between the contribution of salt bridges and the electrostatic interactions,hydrogen bonds,and the dihedral angle conformation of sidechain,while there was weak correlation between the energetic contribution and the formation probability of water-mediated salt bridges.(3)An isolated complex salt bridge in host heterotrimer abc was selected for the combined mutation of K,R,D,E,P,O,and A residues to construct the thermal stability scoring parameters of the collagen.Score and fit the sequences of collagen homotrimers and heterotrimers with known Tm values in previous studies to obtain the collagen thermal stability scoring function,and the local thermal stability of natural type I collagen was predicted.(4)A collagen rational design strategy was proposed.The collagen-like polypeptides with long sequences were split into several short peptides.The constructed collagen scoring function was used to circle and score the short peptides,aiming to screen the unstable combinations.The short peptide sequences were then "linked" as the target peptides with ideal length.The design strategy adopted a computer-aided design method with positive and negative design components and could control the stability and specificity of the target state simultaneously.Using this design strategy,a novel collagen heterotrimer ABC with thermal stability and single composition was designed successfully.