Effects Of Non-Standard Triplet On The Thermal Stability Of Collagen

Collagen is a matrix protein necessary for various tissues and functions in animals.It is widely used in the field of biomaterials.Fibrous collagen has a conserved Gly-X-Y sequence called the standard triplet.There are many non-standard triplets in non-fibrous collagen sequence.Such triplets will cause pathological mutations in fibrous collagen.This study combines experiments and molecular dynamics simulations,analyze the mechanism of non-standard triplets in fibrous and non-fibrous collagens affecting their structures and properties,and provides a theoretical reference for the sequence design and biosynthesis of collagen.Here,collagen-like peptides and recombinant collagen are used as the host models,construct a series of guest collagen models with adjustable mutation numbers by introducing osteogenesis imperfecta mutation sites and natural interruption sequences.Combining molecular dynamics simulation and thermal stability characterization,analyze the unfolding mechanism of collagen,explore the local and overall impact of mutations,and predict the thermal stability of the helix by using local hydrogen bond forces.At the same time,the mutation model was introduced into recombinant collagen for in vitro expression,and collagen molecules with non-standard triplets were built to study the effects of mutations and interrupted sequences on long-chain collagen.The main contents are as follows（1）Combining experiments and simulations to analyze the disruptive effect of a non-standard triplet（Gly→Ala）on fibrous collagen structure.Based on a heterotrimeric model（abc）,7 mutants were constructed by introducing Gly to Ala mutations into each of the 3 chains.The results of CD and DSC showed that the T_m value of the single point mutant decreased by 15°C,while double point mutant decreased by more than 25°C.The triple point mutant did not form a triple helix structure.Probability of hydrogen bond and salt bridge was extract from the molecular dynamics trajectory.Result shows that the force at the mutation site is disturbed.A mathematic function was successfully constructed to show that the product of main-chain hydrogen bonding probabilities is highly correlated with thermal stability（R²=0.92）.Step parameters near the mutation site change,indicating that the triple helix structure is locally unfolded.There is a high correlation between hydrogen bond energy and structural deformation fraction（R²=0.76）,indicating that mutations not only destroy the hydrogen bond force,but also lead to changes in the molecular bending and motion state.（2）Combining experiments and simulations to analyze the damage effect of four non-standard trions with different side chain lengths and disease phenotypes on fibrillar collagen structure.Amino acids with different side chain lengths were introduced into the heterotrimeric model（abc）to construct 12 mutants.Study the effect of chain-specific mutations on the folding state of collagen,analyze the side chain movement mode,and obtain the local hydrogen bond energy threshold during collagen folding.CD results showed that the T_m of the Ser mutant was maintained at around 16°C,while other mutations would lead to unfolding of the collagen triple helix.Molecular dynamics simulation showed that the reduced stability of the mutant was mainly caused by the disruption of backbone hydrogen bonds.Based on the hydrogen bond energy,a local hydrogen bond energy threshold of-10.5 kcal·mol^-1 for predicting triple helix folding or unfolding was obtained.Especially,Val caused the unfolding of triple helices while Ser with a similar side-chain length did not.Structural details suggested that the side-chain hydroxyl group in Ser forms hydrogen bond with the backbone,thus compensating for the mutants’decreased stability.（3）Simulations resolve the effect of a non-standard triplet（G5G natural interruption）similar to the Gly→Ala mutation on non-fibrous collagen structure.The G5G natural interruption and the similar Ala mutation were introduced into（POG）₉ to construct four mutants.Normal dynamic simulations observed that the G5G natural interruption had more main-chain hydrogen bond disruption than the Ala mutation,and a CH···O weak hydrogen bond was found in the G5G interruption sequence,dihedral angle information shows that the G5G model can present a more flexible conformation while maintaining the collagen structure.The heating dynamic simulations of the model was carried out and thermal stability difference consistent with the experiment was observed.The unfolding temperature of the G5G natural interruption was 50 K higher than that of the Ala mutation.The refolding simulation of the unfolded structure showed that Ala in the G5G natural interruption sequence could promote the folding of the collagen structure.（4）Using recombinant collagen as a host model to study the effects of non-standard triplets such as Gly mutations and G5G natural interruption on collagen structure.The bacterial collagen V-ABC derived from S.pyogenes was used as the host model,and the（PPG）₁₀sequence containing the G5G natural interruption and the Gly mutation was introduced into its C-terminus to construct a recombinant expression system,and the fermentation conditions were optimized.The expressed recombinant collagen was characterized for thermal stability.The results showed that the expression of recombinant collagen was mainly influenced by temperature,and that changing the temperature of segmental induction would increase the product.Circular dichroic chromatography results showed that the T_m value of V-ABC-PPG protein was 38.4°C,and the T_m value of collagen with G5G natural interruption was about 37°C.Collagens with Gly mutations had T_m values ranging from 33.4°C to 36.5°C,indicating that natural disruption of G5G had minor effect on the structure of recombinant collagen.