Investigating the Scaling Behavior of Multidomain Proteins under Force using Single Molecule and Ensemble Force-Clamp Spectroscopy

Abstract

A large number of proteins operating under force in vivo are segregated into multidomains, and integrate the unfolding of some of these domains into their mechanical response. Due to the vectorial nature of the force, these proteins are best studied with force spectroscopy techniques. Here we combine single molecule magnetic tweezers with a new technique capable of measuring protein hydrogels using constant force protocols. For our studies we engineer constructs with the B1 domain of protein L, a model alpha-beta protein from Peptostreptococcus magnus. Using magnetic tweezers and HaloTag covalent attachment, we measure the unfolding and refolding of protein L in various force and solutions regimes. At single molecule level, domain unfolding is measured as step-increments in end-to-end length. The measured extensions and unfolding rates as a function of force allow us to estimate the energy barrier between the folded and unfolded states to ∼12 kT and validate the of the use worm-like chain model to describe the step size as a function of force, as previously reported by J. Valle-Orero et al, BBRC 460 (2015). Crosslinking multidomain protein L constructs engineered with eight repeats results in protein hydrogels, which are a few mm long and experience mN forces while extending several microns. We find that hydrogels based on protein L show at constant force a non-exponential smooth response to force and the presence of chemical denaturants or osmolytes have a significant effect on their elasticity. We propose a 3D model to explain the measured behavior of these protein hydrogels which is based on the free energy of a single protein domain under force and which assumes a random orientation and placement of the molecules inside the gel. This model reproduces qualitatively the measured force-clamp response of protein hydrogels and is the first step toward understanding the transition from the probabilistic response of single molecules to the deterministic response that characterizes the functioning of tissues and organs.