Study On Adaptation Mechanism Of Psychrophilic Serine Proteases Using Computational Biology Methods

Psychrophilic enzymes can remain a high catalytic activity at low temperatures,but what are the molecular mechanisms to ensure a relatively high catalytic efficiency of psychrophilic enzymes when their atomic thermal motions have been greatly reduced at low temperatures?Such a special nature of psychrophilic enzyme has been a subject of intense investigation in the fields of enzymology and protein engineering.In order to shed light on the cold-adaptation mechanism of the psychrophilic serine protease,this thesis perform a comparative investigation,using computer simulation methods of the high-temperature molecular dynamics(MD)simulation and the parallel tempering in well-tempered ensemble(PT-WTE),on differences in stability,conformatonal flexibility,and free energy landsacpe(FEL)between the psychrophilic serine protease VPR and its mesophilic homologous counterpart,serine protease proteinase K.The main contents of this thesis are as follows:1.Comparative thermal unfolding study of the psychrophilic and mesophilic serine proteases by high-temperature molecular dynamics simulations.In this section,both crystallographic structures of the psychrophilic VPR and mesophilic PRK were subjected to MD simulations at high temperatures(373 K,473 K,and 573 K)and at room temperature(300 K)as a control.Based on the obtained MD trajectories,the differences in geometrical properties.conformational flexibility,secondary structure,and unfolding path between these two proteases were compared and analyzed.The results show that:i)when the simulation temperature is above 300 K,VPR exhibits greater structural fluctuations,more unstable secondary structures,and higher global conformational flexibility than PRK;ii)despite the similar unfolding path observed for these two proteases,the temperature to initiate unfolding is lower for the psychrophilic VPR(373 K)than for the mesophilic PRK(473 K),and at the same high temperatures,the psychrophilic VPR exhibits unfolding rates faster than those of the mesophilic PRK;?)in simulations under the three high-temperature conditions.VPR shows larger radius of gyration,less numbers of intramolecular hydrogen bond and of close interatomic contacts,more number of protein-solvent hydrogen bond than PRK.and at the same time,the psychrophilic VPR exposes more polar surface area and buries less non-polar area than the mesophilic PRK.The above results lead us to speculate that the lower stability(or higher conformational flexibility)of the psychrophilic VPR most likely originates from its less intramolecular noncovalent interactions(enthalpy effect)and more intermolecular(protein-solvent)interactions,while the higher stability of the mesophilic PRK could be attributed to its more intramolecular noncovalent interactions and more compact hydrophobic packing.In addition,the structural factors responsible for differences in the local conformational flexibility between these two proteases were also analyzed in detail through evaluating the dynamic behavior of the structural environment of the differentiated amino acid residues located at the structurally equivalent positions of the two proteases.The results show that the differences in spatial distributions of disulfide bonds,Ca2+ binding sites,and salt bridges between these two proteases,as well as the changes in enthalpic forces and entropy effect arising from changes in amino acid residues between these two proteases should be crucial factors responsible for the observed different local conformational flexibility of these two proteases.Taken together,results in this section make clear the differences in thermostability and conformational flexibility between the psychrophilic VPR and mesophilic PRK,and,moreover,the structural/physicochemical factors causing these differences were also identified and elucidated.2,Comparative study of psychrophilic and mesophilic serine proteases by using enchanced sampling MD simulations of PT-WTE.In this section,our research subjects,the mesophilic PRK and psychrophilic VPR were subjected to the essential dynamics(ED)analysis to investigate their dynamical structural properties and dynamic personalities,and this was further followed by rebuilding the free energy landscapes(FEL)of these two proteases with the eigenvectors 1(PCI)and 2(PC2)being selected as the reaction coordinates.The two main results obtained are:i)the result of ED analysis reveals that VPR experienced more significant large-scale concerted motions than PRK during simulations.Furthermore,the most pronounced conformational changes causing by the large-scale concerted motion are localized at the N-and C-termini,the loop regions,and the substrate binding region in both proteases.Out of them the substrate-binding region was observed to have more drastic concerted motions in VPR than in PRK,which could be related to the higher catalytic efficiency of VPR,possibly due to the improved substrate affinity arising from the increased concerted fluctuations of the substrate-binding region in VPR;ii)the comparison between FELs of these two proteases reveals that the VPR's FEL has a higher global minimum free energy level,a wider width,and a more rugged free energy surface than PRK's FEL,thus implying that the psychrophilic VPR has a lower thermal stability,greater conformational entropy,and richer conformational diversity as compared to the mesophilic PRK.We speculate the higher flexibility and richer conformational states/substates of VPR could not only improve the substrate affinity but also facilitate the conformational conversion to reaction intermediates during the process of catalytic reaction,thus explaining the higher catalytic efficiency of VPR at low the temperature as observed in biochemical experiments.In conclusion,our computer simulation results reveal that,when compared to the mesophilic PRK,the psychrophilic VPR has lower stability and higher conformational flexibility,and its FEL is characterized by a higher global free energy minimum level,wider width,and more rugged free energy surface.On the one hand,the increased flexibility of the psychrophilic protease is beneficial to the conformational transition.thus producincg,more conformational states/substates that would facilitate the binding of the substrate to the enzyme;on the other hand,the improved flexibility of the psychrophilic VPR could also facilitate enzyme conformational conversion to the reaction intermediate and,as thus would help maintain the its high catalytic efficiency at low temperatures.Our results shed light on the molecular mechanism underlying the stability-flexibility-activity relationship of the psychrophilic serine protease,help facilitate an in-depth understanding of the cold-adaptation mechanism of the psychrophilic enzyme,and may also lay the fundation for further experimental studies.