Anomalous Force-Dependent Transition Rates Unveil Dual Pathways in Folding and Unfolding Dynamics of Acyl-coenzyme A Binding Protein

All-α proteins typically fold rapidly and are unable to withstand high forces. Acyl-coenzyme A binding protein (ACBP), a four-α-helix bundle protein, serves as a model protein for studying the folding dynamics of all-α proteins. In previous biochemistry and single molecule force spectroscopy experiments, a controversy exists for the folding pathway and the conformation of the transition state. In this article, we investigate the folding and unfolding dynamics of ACBP in a force range of 4–10 pN using magnetic tweezers, revealing anomalous force-dependent transition rates. The unfolding rate of ACBP remains nearly constant when force is below 6 pN, and it increases sharply when the force exceeds this threshold, while the logarithm of its folding rate is almost a linear function of force. Detailed analysis combined with molecular dynamics simulations indicates that ACBP has two transition pathways: one dominating at zero or low force and the other dominating at high force. Our results provide strong evidence that stretching force not only modulates the folding and unfolding rates but also switches the transition pathways, leading to complex force response behaviors.