While single-domain proteins often exhibit two-state behavior,some proteins demonstrate complex folding pathways with intermediate states.Here,we investigate force-induced conformational transitions of RNase H using m...While single-domain proteins often exhibit two-state behavior,some proteins demonstrate complex folding pathways with intermediate states.Here,we investigate force-induced conformational transitions of RNase H using magnetic tweezers and reveal structurally distinct intermediate states in the folding and unfolding processes.Inspired by molecular dynamics simulations and verified by experiments on truncations,we found that the unfolding intermediate observed at forces above 8 pN represents a partially unfolded state with the C-terminal helix detached,while the folding intermediate observed below 6 pN corresponds to a partially folded state with only the core region formed,which is not the randomly collapsed molten globule state.Despite these structural differences,our comprehensive kinetic analysis demonstrates that both intermediates can be integrated into a unified quantitative free energy landscape along a single transition pathway.This reconciliation of competing folding intermediates provides fundamental insights into the complex dynamic behaviors of proteins in which different intermediate states are observed.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.12474200,12174322 to HC,32541047,32271367,12204389 to SL)the 111 project(Grant No.B16029)the Research Fund of Wenzhou Institute(Grant No.WIUCASQD2021008)。
文摘While single-domain proteins often exhibit two-state behavior,some proteins demonstrate complex folding pathways with intermediate states.Here,we investigate force-induced conformational transitions of RNase H using magnetic tweezers and reveal structurally distinct intermediate states in the folding and unfolding processes.Inspired by molecular dynamics simulations and verified by experiments on truncations,we found that the unfolding intermediate observed at forces above 8 pN represents a partially unfolded state with the C-terminal helix detached,while the folding intermediate observed below 6 pN corresponds to a partially folded state with only the core region formed,which is not the randomly collapsed molten globule state.Despite these structural differences,our comprehensive kinetic analysis demonstrates that both intermediates can be integrated into a unified quantitative free energy landscape along a single transition pathway.This reconciliation of competing folding intermediates provides fundamental insights into the complex dynamic behaviors of proteins in which different intermediate states are observed.
