Mapping the Energy Landscape for Second Stage Folding of a Single Membrane Protein

Abstract

Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, E. coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (delta G = 6.5 kT) but a large unfolding barrier (21.3 kT) that can maintain the protein in a folded state for long periods of time (t1/2 ∼ 3.5 hrs). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure.