Connecting Conformational Stiffness of the Protein With Energy Landscape by a Single Experiment

Abstract

Proteins are versatile biopolymers whose functions are determined by their structures. Understanding the structural dynamicity, with respect to energy landscape, is essential to describe their biological functions. The ability to study the dynamical properties of a single protein molecule is thus crucial, but ensuring that multiple physical properties can be simultaneously extracted within a single experiment on the exact same molecule in real time has hitherto been infeasible.Here, we present microfluidics-magnetic tweezers technology that surmounts this limitation, providing real-time dynamic information about changes in the structural properties of a single protein molecule, even as it encounters a changing physicochemical environment. We illustrate the versatility of the method by studying electrolyte-dependent conformational flexibility and the energy landscape of substrate protein L under force. Changing salt concentrations reshapes the energy landscape by two specific ways: it speeds-up refolding kinetics while slowing down unfolding kinetics. From the same trajectory, we calculate the stiffness of the protein polymer, a quantity that varies with salt concentration. The data is described within the framework of a modified ‘electrolyte FJC model’ that we propose and study here. The observed correlation between ∆G, kinetics and polymer elasticity connects protein chain physics and the energy landscape, while the experimental methodology we describe and benchmark should have wide-ranging applications in single-molecule experiments.