| Hyperthermophilic proteins are a class of unique proteins isolated from thermophilic microorganisms that survive in extreme environments such as deep-sea hydrothermal vents and hot springs.These proteins have received significant attention due to their stability and specific activity at high temperatures.Studying the effect of temperature on the structure and function of hyperthermophilic proteins is of great scientific and practical significance.From the perspective of protein stabilization,such research can reveal the changes in the structure and function of proteins at different temperatures,helping to address stability issues in disease occurrence and industrial applications of proteins.From the perspective of enzyme engineering and biotechnology,this research can elucidate the high-temperature activation mechanism of hyperthermophilic proteins,aiding in the modification and design of normal proteins to improve industrial process efficiency and yield.In summary,studying the effect of temperature on the structure and function of hyperthermophilic proteins can enhance our understanding of protein stability and function,promote the development of life sciences,and have important implications for technological innovation in biotechnology and industrial production.In recent decades,experimental techniques have made significant progress in directly measuring and validating the structure and function of hyperthermophilic proteins,limitations such as the high cost of high-temperature equipment,limited protein acquisition channels,and certain difficulties in structure resolution still need to be overcome in experimental research.With the rapid development of computer technology,computational simulation methods have certain advantages in studying the effect of temperature on the structure and function of hyperthermophilic proteins.They do not require high-temperature experimental instruments,have flexible and controllable simulation processes,explore various structures and conformations,and can reflect the dynamic behavior and interactions of proteins.This paper focuses on the functions of protecting nucleic acids,catalyzing and cleaving nucleic acids.It mainly uses a research strategy that combines molecular dynamics and accelerated molecular dynamics simulations to examine the relationship between temperature,thermal stability and activity of hyperthermophilic proteins.The specific research contents are as follows:1.Theoretical study of the mechanism of DNA protection by hyperthermophilic protein Ape10b2 at high temperaturesIn this study,the structural stability and interaction details of DNA binding to Ape10b2 protein at four temperatures(300 K,343 K,363 K,and 373 K)were investigated by using a combination of multiple short molecular dynamics simulations and binding free energy calculations.The results show that DNA binds to Ape10b2 in a “spanning” mode.At high temperatures(343 K,363 K,and 373 K),the α2 and loop5 regions of the Ape10b2 protein are tightly bound to DNA through hydrogen bonding,which stabilizes the DNA double helix in the B conformation,and serves to protect the nucleic acid.Arg42 and Arg46 on α2 and Arg86 on loop5 are the key residues of Ape10b2 to stabilize DNA at high temperatures.The above research results can help to understand the mechanism of Ape10b2 protecting DNA at high temperatures in the molecular level,and provide important theoretical clues for research on the protection of nucleic acids by hyperthermophilic proteins.2.Theoretical study on the effect of temperature on the relationship between the dimerization process and catalytic function of hyperthermophilic esterase Pf2001In this work,based on the inference proposed in experimental studies that Pf2001 can only exert its catalytic function in the dimeric state,a research strategy combining classical molecular dynamics simulations and accelerated molecular dynamics simulations was used at different temperatures.We have studied the dynamic process of Pf2001 dimerization that is difficult to capture in experiment,and determined the“preparatory state” monomer conformation that is conducive to the dimerization of Pf2001 at high temperatures.The significant reduction in electrostatic repulsion at the dimer interface is the key factor in dimerization at high temperatures.There is a “tictac-toe” dimerization interface during the formation of the dimeric state.The π-πinteractions between the residues(Phe188,Trp194,Phe198,and Phe202)at this interface are the main factors in the formation of the catalytic pocket.α-helix induced rearrangement of the cap domain is an important step in promoting the formation of the“tic-tac-toe” dimerization interface.The above research results will help us deeply understand the catalytic mechanism of temperature-dependent hyperthermophilic esterases at the molecular level,and provide a reliable theoretical basis for the transformation of ordinary enzymes and their application in industrial production in extreme environments.3.Theoretical study on the effect of temperature on the relationship between structural dynamics and nucleic acid cleavage function of hyperthermophilic protein MjAgoIn this study,a research strategy combining homology modeling,molecular docking,molecular dynamics simulations,and binding free energy calculations was employed to investigate the effects of conformational changes of MjAgo at different temperatures on DNA binding and its cleavage activity.The results show that MjAgo with locally flexible conformation undergoes a conformational transition that leads to a tighter binding with DNA by adjusting the width of the nucleic acid binding channel and increasing the affinity of MjAgo-ds DNA binding at physiological temperature(i.e.high temperature 358 K).In addition,we also found that the local flexible transition of the protein conformation induced the residues Tyr194,His213,Tyr258,Met259,Tyr442,and His685 to form hydrogen bonds with DNA only at 358 K,presenting a unique“high-temperature binding” characteristic,which is beneficial to MjAgo binding to DNA enriches the understanding of Tyr194 and His213 as key residues affecting MjAgo cleavage activity.The above research results help us gain a deeper understanding of the conformational transition strategy adopted by MjAgo to exert its cleavage activity at physiological temperature,and can provide valuable theoretical reference for the design of highly active hyperthermophilic Ago proteins. |
