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

TL;DR

This study investigates the force-dependent folding and unfolding dynamics of the two-state protein Acylphosphatase, revealing a complex free energy landscape shaped by its large contact order and providing new insights into its mechanical properties.

Contribution

The paper presents the first measurements of AcP's folding and unfolding rates at low forces using magnetic tweezers, uncovering a two-barrier free energy landscape influenced by contact order.

Findings

Unfolding rates vary with force, showing different sensitivities below and above ~27 pN.

The free energy landscape has two energy barriers, indicating a complex folding pathway.

Large contact order contributes to a high energy barrier at low forces.

Abstract

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.