Probing Single-Molecule Protein Spontaneous Folding–Unfolding Conformational Fluctuation Dynamics: The Multiple-State and Multiple-Pathway Energy Landscape

Abstract

Protein conformational dynamics often plays a critical role in protein functions. We have characterized the spontaneous folding–unfolding conformational fluctuation dynamics of calmodulin (CaM) at thermodynamic equilibrium conditions by using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy. We have identified multiple folding transition pathways and characterized the underlying energy landscape of the single-molecule protein conformational fluctuation trajectories, using a model analysis based on the detailed balance rate process principle. Our results suggest that the folding dynamics of CaM molecules involves a complex multiple-pathway multiple-state energy landscape, rather than an energy landscape of two-state dynamical process. Our probing single-molecule FRET fluctuation experiments demonstrate a new approach of studying spontaneous protein folding–unfolding conformational dynamics at the equilibrium that features recording long time single-molecule conformational fluctuation trajectories