Studying Membrane Protein Thermodynamics Using a Steric Trap

Abstract

An understanding of the molecular forces that specify a protein's structure is essential for many of the central quests of structural biology. While there has been a larger effort to understand soluble protein folding, we know very little about membrane protein folding energetics. We are developing a novel method to study the forces that stabilize membrane proteins in lipid bilayers, which we termed the ‘Steric Trap method.’ The steric trap method couples protein unfolding to a measurable binding event by exploiting steric repulsion and the high affinity of the streptavidin/biotin interaction. To do this, we introduce two biotin tags on a target protein that are close in space and employ monovalent streptavidin (mSA) as our steric trap. A single mSA can bind without steric hindrance to the folded protein, but a second mSA can only bind when the protein unfolds due to steric overlap. Thus, the binding affinity of the second streptavidin provides a measure of unfolding free energy because binding is coupled to unfolding. We have developed this method on the water-soluble protein, dihydrofolate reductase (DHFR). When two biotin-labeling sites were rationally designed to be close to one another in space and near the enzyme active site of DHFR, enzymatic activity was reversibly abolished upon incubation with a molar excess of monovalent streptavidin. Incubation with a stabilizing ligand shifted streptavidin binding curves, confirming that the steric trapping can quantitatively detect changes in protein stability. Results on our application of the steric trap method to membrane proteins will be presented. The steric trap method could be a powerful tool for measuring protein association affinities, studying unfolding energetics and investigating membrane protein unfolded states in the context of membrane environments.