Membrane protein folding makes the transition.

T he study of the folding of membrane proteins has lagged far behind that of small soluble proteins—yet proteins that reside within biological membranes account for approximately a third of all proteomes. The article by Huysmans et al. in this issue of PNAS (1) represents a breakthrough by reporting a comprehensive φ-value analysis of the folding of a membrane protein (i.e., PagP) into a lipid bilayer. φ-value analysis is the most powerful tool for experimental analysis of protein folding pathways (2). It combines protein engineering with equilibrium and kinetic measurements to determine which regions of a protein are largely folded (high φ-values) or largely unfolded (low φ-values) at the rate-limiting transition state. There are two major structural classes of integral membrane proteins: α-helical bundles and β-barrels. The latter are found in the outer membranes of Gramnegative bacteria and mitochondria, whereas helical structures are ubiquitous, occurring, for example, in plasma and inner membranes. PagP has a β-barrel structure and resides in the outer membrane of Escherichia coli. The analysis presented by Huysmans et al. (1) sheds light on the transition state structure for formation of this β-barrel in a bilayer. The work complements a previous φ-value study of membrane protein folding that was the first to map out a membrane protein transition state, but in this case, for folding of an α-helical protein, bacteriorhodopsin, into lipid-detergent mixtures (3). Thus detailed insight into the folding mechanisms of the two major membrane protein structural classes is now emerging. A key feature of this current study (1) is the examination of folding into a lipid bilayer. To achieve this, Huysmans et al. (1) exploited a previously demonstrated trait of β-barrel proteins: they can be reversibly refolded from a urea-denatured state into lipid bilayer vesicles, and the kinetics and thermodynamics of the folding reaction can be determined (4, 5). The successful demonstration of this for PagP (6) paved the way for a φ-value study. Identification of conditions for reversible folding of PagP relied on manipulation of the lipid to protein ratio. At low ratios, PagP folding was protein concentration-dependent, but at the lipid-to-protein ratio used here (3,200:1), refolding was completely reversible and protein concentration– independent. The folding and unfolding kinetics were determined, and, perhaps surprisingly, conditions could be found (urea concentrations greater than approximately 8 M) in which folding could be characterized as a simple two-state reaction. It is also of note that the choice of a β-barrel protein means that folding can be studied from an apparently fully unfolded, denatured state. Most helical membrane proteins retain significant residual structure in common denaturants such as urea and SDS, but the denatured state of PagP in 10 M urea, although associated with the bilayer, is essentially unstructured (as judged by circular dichroism and fluorescence spectra). The use of urea gives added importance to the PagP work, as urea apparently does not partition into the bilayer, unlike SDS used for studies of the folding of several α-helical proteins. Nineteen variants of PagP were investigated, at least one mutation in each β-strand, thus giving a snapshot across the entire protein. The protein folds via a polarized transition state with the barrel partly formed and the C-terminal part of the protein significantly more structured than the N-terminal regions. The authors propose that the results are consistent with a tilted folding-insertion mechanism (Fig. 1), as has been seen in simulations for insertion of folded OmpA into a bilayer (7). The results also agree with previously proposed models of concerted folding and insertion of β-barrels into membranes (8); as an unfolded barrel protein within a bilayer is unlikely, as it would expose potential hydrogen bonding groups (9). Helical membrane proteins, by contrast, seem to fold by a fundamentally different mechanism than barrels (9, 10). Individual transmembrane helices can be stable entities in bilayers, as backbone hydrogen bonding is satisfied locally within the helix. Fig. 1. Schematic diagrams of proposed folding models for β-barrel and α-helical membrane proteins, highlighting potential transition state structures from φ-value studies. Folding of a β-barrel protein occurs from a fully denatured, membrane-absorbed state in urea with a tilted, partly inserted transition state as proposed by Huysmans et al. (1). In contrast, folding of an α-helical protein such as bacteriorhodopsin occurs from a partly denatured state in SDS, which contains some helical content. The transition state is proposed to have significant native helix content. Only one transmembrane helix has been analyzed by φ-values (3), and this suggests a largely formed helix with partial helix formation at the cytoplasmic side (shown in the bottom of this diagram, with this helix outlined in black). The degree of structure in other helices is estimated from previous studies (e.g., ref 15). Unfolded structure is shown in red and folded structure in blue. SDS is shown in green. PagP was folded into lipid bilayers vesicles, whereas bacteriorhodopsin was folded into mixed DMPC/CHAPS micelles (the detergent CHAPS is shown here capping the edges of a DMPC disc).