Kinetics of Insertion and Folding of Outer Membrane Proteins by Gel Electrophoresis

To examine the mechanisms of folding and insertion of TMPs into membranes, kinetic studies are instrumental, for example, for the analysis of folding steps and involved intermediates or for the determination of activation energies. For many β-barrel transmembrane proteins (β-TMPs) it has been shown that the folded, functional form can be separated from the unfolded form by a simple electrophoretic mobility assay. The only requirements for a separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are that the folded form is sufficiently stable and that the samples are not heat-denatured before the electrophoresis is performed. Many folded β-TMPs resist the treatment with SDS at room temperature and are stable against forces during electrophoresis. On the other side, SDS also binds to unfolded forms of β-TMPs and 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. At selected times, samples are taken from the reaction. In these samples, folding is stopped by addition of SDS. At the end of the entire folding reaction, all samples are analyzed by SDS-PAGE and the fractions of folded β-TMP that they contain are determined by densitometry.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 chaperones) 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.