The Formation of ß-Strand Nine (ß9) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers.

Transmembrane proteins span lipid bilayer membranes and serve essential functions in all living cells. Membrane-inserted domains are of either α-helical or ß-barrel structure. Despite their biological importance, the biophysical mechanisms of the folding and insertion of proteins into membranes are not well understood. While the relative composition of the secondary structure has been examined by circular dichroism spectroscopy in folding studies for several outer membrane proteins, it is currently not known how individual ß-strands fold. Here, the folding and insertion of the ß-barrel assembly machinery protein A (BamA) from the outer membrane of Escherichia coli into lipid bilayers were investigated, and the formation of strand nine (ß9) of BamA was examined. Eight single-cysteine mutants of BamA were overexpressed and isolated in unfolded form in 8 M urea. In each of these mutants, one of the residues of strand ß9, from R572 to V579, was replaced by a cysteine and labeled with the fluorophore IAEDANS for site-directed fluorescence spectroscopy. Upon urea-dilution, the mutants folded into the native structure and were inserted into lipid bilayers of dilauroylphosphatidylcholine, similar to wild-type BamA. An aqueous and a membrane-adsorbed folding intermediate of BamA could be identified by strong shifts in the intensity maxima of the IAEDANS fluorescence of the labeled mutants of BamA towards shorter wavelengths, even in the absence of lipid bilayers. The shifts were greatest for membrane-adsorbed mutants and smaller for the inserted, folded mutants or the aqueous intermediates. The spectra of the mutants V573C-, L575C-, G577C-, and V579C-BamA, facing the lipid bilayer, displayed stronger shifts than the spectra recorded for the mutants R572C-, N574C-, T576C-, and K578C-BamA, facing the ß-barrel lumen, in both the membrane-adsorbed form and the folded, inserted form. This alternating pattern was neither observed for the IAEDANS spectra of the unfolded forms nor for the water-collapsed forms, indicating that strand ß9 forms in a membrane-adsorbed folding intermediate of BamA. The combination of cysteine scanning mutagenesis and site-directed fluorescence labeling is shown to be a valuable tool in examining the local secondary structure formation of transmembrane proteins.

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 ß-barrel transmembrane proteins (ß-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 ß-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 ß-TMPs and prevents their folding into ß-barrel structure. These observations have been used to develop a simple assay to monitor the kinetics of ß-barrel tertiary structure formation in a membrane environment by electrophoresis. A folding reaction of a ß-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 ß-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 ß-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 ß-TMPs.