Single-molecule magnetic tweezers(MTs) have revealed multiple transition barriers along the unfolding pathway of several two-state proteins, such as GB1 and Csp. In this study, we utilized MTs to measure the force-dep...Single-molecule magnetic tweezers(MTs) have revealed multiple transition barriers along the unfolding pathway of several two-state proteins, such as GB1 and Csp. In this study, we utilized MTs to measure the force-dependent folding and unfolding rates of both protein L(PLWT) and its Y47W mutant(PLY47W) where the mutation point is not at the force-bearing β-strands. The measurements were conducted within a force range of 3–120 pN. Notably, the unfolding rates of both PLWT and PWY47W exhibit distinct force sensitivities below 50 pN and above 60 pN, implying a two-barrier free energy landscape. Both PLWT and PLY47W share the same force-dependent folding rate and the same transition barriers,but the unfolding rate of PLY47W is faster than that of PLWT. Our finding demonstrates that the residue outside of the force-bearing region will also affect the force-induced unfolding dynamics.展开更多
Enzymes are the major players for many biological processes.Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble leve...Enzymes are the major players for many biological processes.Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level.Yet,these single-molecule experiments are technically di±cult and generally require complicated experimental design.Here,we develop a Holliday junction(HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET(sm-FRET).We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events.Thanks to the multiple-arms structure of HJ,the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm.Therefore,it is possible to independently change the substrate arm to study different enzymes with similar functions.Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations.Moreover,this method can be easily extended to study other types of DNA-binding enzymes without too much modi fication of the design.We anticipate it can find broad applications in single-molecule enzymology.展开更多
Understanding the processes of protein adsorption/desorption on nanoparticles' surfaces is important for the development of new nanotechnology involving biomaterials; however, an atomistic resolution picture for t...Understanding the processes of protein adsorption/desorption on nanoparticles' surfaces is important for the development of new nanotechnology involving biomaterials; however, an atomistic resolution picture for these processes and for the simultaneous protein conformational change is missing. Here, we report the adsorption of protein GB1 on a polystyrene nanoparticle surface using atomistic molecular dynamic simulations. Enabled by metadynamics, we explored the relevant phase space and identified three protein states, each involving both the adsorbed and desorbed modes. We also studied the change of the secondary and tertiary structures of GB1 during adsorption and the dominant interactions between the protein and surface in different adsorption stages. The results we obtained from simulation were found to be more adequate and complete than the previous one. We believe the model presented in this paper, in comparison with the previous ones, is a better theoretical model to understand and explain the experimental results.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.12174322 to HC and 12204124 to ZG)111 Project(Grant No.B16029)+1 种基金the Graduate Scientific Research Foundation of Wenzhou University(Grant No.3162023003034 to JH)research grant from Wenzhou Institute。
文摘Single-molecule magnetic tweezers(MTs) have revealed multiple transition barriers along the unfolding pathway of several two-state proteins, such as GB1 and Csp. In this study, we utilized MTs to measure the force-dependent folding and unfolding rates of both protein L(PLWT) and its Y47W mutant(PLY47W) where the mutation point is not at the force-bearing β-strands. The measurements were conducted within a force range of 3–120 pN. Notably, the unfolding rates of both PLWT and PWY47W exhibit distinct force sensitivities below 50 pN and above 60 pN, implying a two-barrier free energy landscape. Both PLWT and PLY47W share the same force-dependent folding rate and the same transition barriers,but the unfolding rate of PLY47W is faster than that of PLWT. Our finding demonstrates that the residue outside of the force-bearing region will also affect the force-induced unfolding dynamics.
基金The authors greatly appreciate the-nancial support from National Natural Science Foundation of China(Grant Nos.21522402,11674153,11374148,11334004 and 21771103)the Fundamental Research Funds for the Central Universities(Nos.020414380070,020414380050 and 020414380058)Natural Science Foundation of Jiangsu Province(No.BK20160639)and the Shuangchuang Program of Jiangsu Province.
文摘Enzymes are the major players for many biological processes.Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level.Yet,these single-molecule experiments are technically di±cult and generally require complicated experimental design.Here,we develop a Holliday junction(HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET(sm-FRET).We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events.Thanks to the multiple-arms structure of HJ,the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm.Therefore,it is possible to independently change the substrate arm to study different enzymes with similar functions.Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations.Moreover,this method can be easily extended to study other types of DNA-binding enzymes without too much modi fication of the design.We anticipate it can find broad applications in single-molecule enzymology.
基金supported by the National Natural Science Foundation of China(Grant Nos.11774158,11274157,31671026,and 11334004)the National Basic Research and Development Program of China(Grant No.2013CB834100)Priority Academic Program Development(PAPD)Project of Jiangsu Higher Education Institutions
文摘Understanding the processes of protein adsorption/desorption on nanoparticles' surfaces is important for the development of new nanotechnology involving biomaterials; however, an atomistic resolution picture for these processes and for the simultaneous protein conformational change is missing. Here, we report the adsorption of protein GB1 on a polystyrene nanoparticle surface using atomistic molecular dynamic simulations. Enabled by metadynamics, we explored the relevant phase space and identified three protein states, each involving both the adsorbed and desorbed modes. We also studied the change of the secondary and tertiary structures of GB1 during adsorption and the dominant interactions between the protein and surface in different adsorption stages. The results we obtained from simulation were found to be more adequate and complete than the previous one. We believe the model presented in this paper, in comparison with the previous ones, is a better theoretical model to understand and explain the experimental results.