Dynamic analysis methods are important for analyzing long simulations such as folding simulations. Relaxation mode analysis, which approximately extracts slow modes and rates, has been applied in molecular dynamics (MD) simulations of protein systems. Previously, we showed that slow modes are suitable for analyzing simulations in which large conformational changes occur. Here, we applied relaxation mode analysis to folding simulations of a designed mutant of protein G, NuG2, to investigate its folding pathways. The folding simulations of NuG2 were previously performed for this mutant with Anton. In the present study, the free energy surfaces were calculated by projecting the coordinates on the axis of the slow relaxation modes obtained from relaxation mode analysis. We classified various characteristic states such as native, nativelike, intermediate, and random states and clarified two main folding pathways. In the early folding process, the first and second β strands formed an N-terminal β-sheet. After the early folding process, the fourth β strand formed along the first β strand in the same or opposite direction as the native structure; two characteristic intermediate states were identified. Finally, the intermediate structures folded to the native structure in the folding process. Relaxation mode analysis can be applied to folding simulations of complex proteins to investigate their folding processes.
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