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
Src SH3 protein domain is a typical two-state protein which has been confirmed by research of denaturant-induced unfolding dynamics.Force spectroscopy experiments by optical tweezers and atomic force microscopy have m...Src SH3 protein domain is a typical two-state protein which has been confirmed by research of denaturant-induced unfolding dynamics.Force spectroscopy experiments by optical tweezers and atomic force microscopy have measured the force-dependent unfolding rates with different kinds of pulling geometry.However,the equilibrium folding and unfolding dynamics at constant forces has not been reported.Here,using stable magnetic tweezers,we performed equilibrium folding and unfolding dynamic measurement and force-jump measurement of src SH3 domain with tethering points at its N-and C-termini.From the obtained force-dependent transition rates,a detailed two-state free energy landscape of src SH3 protein is constructed with quantitative information of folding free energy,transition state barrier height and position,which exemplifies the capability of magnetic tweezers to study protein folding and unfolding dynamics.展开更多
Force spectrum measurements with constant loading rates are widely used in single-molecule manipulation experiments to study the mechanical stability and force response of biomolecules.Force-dependent transition rates...Force spectrum measurements with constant loading rates are widely used in single-molecule manipulation experiments to study the mechanical stability and force response of biomolecules.Force-dependent transition rates can be obtained from the transition force distribution,but it is limited to the force range with non-zero force distribution.Although constant loading rate control can be realized with magnetic tweezers,the loading rate range is limited due to the slow movement of permanent magnets.Non-linear exponential and exponential squared force loading functions are more feasible in magnetic tweezers,while there is no theoretical result available for these two kinds of non-linear force loading functions.In this study,we solved the unfolding process of a protein following Bell's model under nonlinear exponential and exponential squared force loading functions,which offer a broader range of unfolding force distribution compared to the traditional constant loading rate experiments.Furthermore,we derived two force loading functions,which can produce uniform unfolding force distribution.This research contributes fundamental equations for the analysis of experimental data obtained through single-molecule manipulation under nonlinear force loading controls,paving the way for the use of nonlinear force control in magnetic tweezer experiments.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 National Natural Science Foundation of China(Grant Nos.11874309 and 11474237)the 111 Project(Grant No.B16029)。
文摘Src SH3 protein domain is a typical two-state protein which has been confirmed by research of denaturant-induced unfolding dynamics.Force spectroscopy experiments by optical tweezers and atomic force microscopy have measured the force-dependent unfolding rates with different kinds of pulling geometry.However,the equilibrium folding and unfolding dynamics at constant forces has not been reported.Here,using stable magnetic tweezers,we performed equilibrium folding and unfolding dynamic measurement and force-jump measurement of src SH3 domain with tethering points at its N-and C-termini.From the obtained force-dependent transition rates,a detailed two-state free energy landscape of src SH3 protein is constructed with quantitative information of folding free energy,transition state barrier height and position,which exemplifies the capability of magnetic tweezers to study protein folding and unfolding dynamics.
基金supported by the National Natural Science Foundation of China (Grant Nos. 12174322 to HC, 12204124 to ZG, 32271367 and 12204389 to SL)the 111 project (Grant No. B16029)the Research Fund of Wenzhou Institute
文摘Force spectrum measurements with constant loading rates are widely used in single-molecule manipulation experiments to study the mechanical stability and force response of biomolecules.Force-dependent transition rates can be obtained from the transition force distribution,but it is limited to the force range with non-zero force distribution.Although constant loading rate control can be realized with magnetic tweezers,the loading rate range is limited due to the slow movement of permanent magnets.Non-linear exponential and exponential squared force loading functions are more feasible in magnetic tweezers,while there is no theoretical result available for these two kinds of non-linear force loading functions.In this study,we solved the unfolding process of a protein following Bell's model under nonlinear exponential and exponential squared force loading functions,which offer a broader range of unfolding force distribution compared to the traditional constant loading rate experiments.Furthermore,we derived two force loading functions,which can produce uniform unfolding force distribution.This research contributes fundamental equations for the analysis of experimental data obtained through single-molecule manipulation under nonlinear force loading controls,paving the way for the use of nonlinear force control in magnetic tweezer experiments.
