Analysis of the Kinetics of Folding and Insertion of β-Barrel Membrane Proteins by Gel-Electrophoresis.

Investigations on the mechanisms of folding and insertion of transmembrane proteins (TMPs) into lipid bilayers require an analysis of the time course of the structural changes of a TMP. Kinetic studies are essential for the analysis of individual folding steps, for identification of folding intermediates and for obtaining activation energies and the rate-limiting steps of folding. For many β-barrel transmembrane proteins (β-TMPs), it has been shown that the folded, functional form can be separated from the unfolded, nonfunctional form by an electrophoretic mobility assay. The requirements for a separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are: (1) the folded form is sufficiently stable and (2) the samples are not heat-denatured before loading them onto the gel for the electrophoresis. Many folded and lipid-bilayer integrated β-TMPs may be extracted into micelles of SDS, but resist SDS-induced unfolding and the electrophoresis when SDS-PAGE is performed at room temperature or in a cold room. Unfolded β-TMPs bind SDS, which prevents their folding into β-barrel structure. These observations have been used to develop a simple assay to monitor the kinetics of β-barrel tertiary structure formation in a membrane environment by electrophoresis. A folding reaction of a β-TMP is initiated by dilution of the denaturant in the presence of preformed lipid bilayers, proteoliposomes, or membrane vesicles. After selected time intervals, samples are taken from the reaction and mixed with SDS containing Laemmli buffer to prevent further folding. The fraction of folded β-TMPs in each sample is analyzed by SDS-PAGE followed by densitometry of the gels. An advantage of this kinetic assay is that it not only allows a direct determination of fractions of folded and unfolded forms at a selected time during folding of the β-TMP into a membrane, but also facilitates the determination of the impact of folding factors, e.g., molecular chaoperones or folding machinery that most often have a different molecular mass and electrophoretic mobility. The assay has been very useful to examine how folding and insertion is affected by the structure of the phospholipids in the lipid bilayer and how folding machinery compensates for the presence of membrane lipids that retard folding and insertion of β-TMPs.