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Free energy landscape of two-state protein Acylphosphatase with large contact order revealed by force-dependent folding and unfolding dynamics

Acylphosphatase (AcP) is a small protein with 98 amino acid residues that catalyzes the hydrolysis of carboxyl-phosphate bonds. AcP is a typical two-state protein with slow folding rate due to its relatively large contact order in the native structure. The mechanical properties and unfolding behavior of AcP has been studied by atomic force microscope. But the folding and unfolding dynamics at low forces has not been reported. Here using stable magnetic tweezers, we measured the force-dependent folding rates within a force range from 1 pN to 3 pN, and unfolding rates from 15 pN to 40 pN. The obtained unfolding rates show different force sensitivities at forces below and above ~27 pN, which determines a free energy landscape with two energy barriers. Our results indicate that the free energy landscape of small globule proteins have general Bactrian camel shape, and large contact order of the native state produces a high barrier dominate at low forces.