The thermodynamic stabilities of membrane proteins are of fundamental interest to provide a biophysical description of their structure-function relationships because energy determines conformational populations. In addition, structure-energy relationships can be exploited in membrane protein design and in synthetic biology. To determine the thermodynamic stability of a membrane protein, it is not sufficient to be able to unfold and refold the molecule: establishing path independence of this reaction is essential. Here we describe the procedures required to measure and verify path independence for the folding of outer membrane proteins in large unilamellar vesicles.
Keywords: Equilibrium; Folding; Large unilamellar vesicles; Outer membrane protein; PagP; Path independence; Stability; Thermodynamics.