Abstract
Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques. Recently two-state property of Csp was confirmed by atomic force microscopy (AFM) through direct pulling measurement, while several long-lifetime intermediate states were found by force-clamp AFM. We systematically studied force-dependent folding and unfolding dynamics of Csp using magnetic tweezers with intrinsic constant force capability. Here we report that Csp mostly folds and unfolds with a single step over force range from 5 pN to 50 pN, and the unfolding rates show different force sensitivities at forces below and above ~8 pN, which determines a free energy landscape with two barriers and a transient intermediate state between them along one transition pathway. Our results provide a new insight on protein folding mechanism of two-state proteins.
Highlights
Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques
Most small single-domain proteins fold to a unique threedimensional structure, and the thermodynamics of protein folding and unfolding can be described by a two-state model with a highly populated native state and unfolded state only[1,2]
Force-dependent unfolding of Csp measured at constant loading rates
Summary
Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques. We systematically studied force-dependent folding and unfolding dynamics of Csp using magnetic tweezers with intrinsic constant force capability. We report that Csp mostly folds and unfolds with a single step over force range from 5 pN to 50 pN, and the unfolding rates show different force sensitivities at forces below and above ~8 pN, which determines a free energy landscape with two barriers and a transient intermediate state between them along one transition pathway. Force-dependent transition rate and detection of shortlived intermediate states provide hints to study the complex free energy landscape of proteins[8]. We report the full-scale study of force-dependent folding and unfolding dynamics of Csp under constant forces using magnetic tweezers. A free energy landscape with two barriers and a transient intermediate state is constructed based on the experimental results
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