One β Hairpin Follows the Other: Exploring Refolding Pathways and Kinetics of the Transmembrane β‐Barrel Protein OmpG

Abstract

Despite their enormous relevance to cellular vitality, the folding mechanisms of only a few transmembrane proteins have been studied. From these studies, only a handful of bstranded membrane proteins were characterized. Current models describe that transmembrane b barrels fold into the lipid membrane in two major steps. Firstly, the unfolded polypeptide interacts with the lipid surface where it folds, tilts, and then inserts into the membrane. Consequently, it is thought that single b strands and b hairpins form unstable units, and that b-barrel proteins (pre-)fold prior to their insertion into the cellular membrane. Experiments studying the (un-)folding of membrane proteins are conventionally carried out by using thermal or chemical denaturation. In most cases, membrane proteins that were solubilized in detergent and/or exposed to approximately 4–10m urea were studied. In vivo membrane proteins fold under different conditions. Thus, the folding pathways studied may be different from those that occur in nature. Single-molecule force spectroscopy (SMFS) represents a unique approach to studying the refolding of membrane proteins into the lipid membrane. SMFS is used to unfold and refold membrane proteins under conditions typical for their physiological environment such as pH, electrolytes, temperature, and, importantly in the absence of any chemical denaturant or detergent. In such experiments, a single membrane protein is first mechanically unfolded and its polypeptide is fully stretched. Then this unfolded polypeptide is relaxed to allow refolding into the membrane bilayer. Repeated mechanical unfolding is used to determine which structural regions of the membrane protein are refolded. Allowing the polypeptide different refolding times addresses the refolding kinetics of structural regions. Thus, SMFS can be used to detect the mechanical unfolding pathways and the equilibrium refolding pathways of a membrane protein. In previous SMFS work, the mechanical unfolding and refolding of many different water-soluble proteins have been investigated. However, compared to the variety of water-soluble proteins that were characterized, SMFS of membrane proteins reveals much more detailed unfolding and folding pathways. To date, the refolding of b-barrel membrane proteins into a lipid membrane has never been addressed by SMFS. Herein we report the application of SMFS to unfold and refold the outer membrane protein G (OmpG) from Escherichia coli (Figure 1). The structure of OmpG comprises 14 b strands that form a transmembrane b-barrel pore. Six short turns connect individual b strands on the periplasmic side and seven longer loops (L1–L7) on the extracellular side. In vitro experiments show that OmpG is gated by loop L6, which controls the permeability of the pore in a pHdependent manner. In previous SMFS studies, we found that the b barrel of OmpG unfolds via many intermediates. The main unfolding pathway described the stepwise unfolding of single b hairpins. This unfolding pathway was much more detailed than that detected for the water-soluble b-barrel green fluorescent protein (GFP), which mainly unfolds in one step when a sufficiently high pulling force was applied. In our refolding experiments, OmpG that had been reconstituted in native E. coli lipid membranes was first imaged by AFM. Then, the AFM tip was pushed onto the OmpG surface to facilitate the nonspecific attachment of the N terminus (Figure 1). Withdrawal of the AFM tip stretched the terminus and induced the unfolding of OmpG. Force– distance (F–D) curves recorded the force peaks that reflect the unfolding steps of a single OmpG (Figure 1). Each unfolding step represents that of a b hairpin of the transmembrane b barrel. To refold the partially unfolded OmpG, we stopped withdrawal before unfolding the last b hairpin VII. Then, we relaxed the unfolded polypeptide by approaching the AFM tip close to the membrane (ca. 5 nm). After a given time to allow the polypeptide to refold, the protein was unfolded again to probe which structural regions refolded into the lipid membrane (see Figure S1 in the Supporting Information). Individual F–D curves of the refolding polypeptide showed a series of force peaks that varied in occurrence (Figure 1). These force peaks were detected at similar positions as upon initial unfolding of OmpG. If b hairpins had folded without inserting or had attached to themembrane surface, the force peaks would have been detected at shifted positions (see the Supporting Information, Part 2). Similarly, force peaks which are characteristic for the folding of membrane proteins, would have changed their position if misfolding events had occurred. Thus, the unfolded OmpG polypeptide folded and inserted single b hairpins into the native E. coli lipid membrane. Probing the content of refolding in dependence of different refolding times (0.1–5 s) [*] M. Damaghi, Dr. C. A. Bippes, Prof. Dr. D. J. M ller ETH Z rich, Dept. of Biosystems Science and Engineering 4058 Basel (Switzerland) Fax: (+41)61-387-3994 E-mail: daniel.mueller@bsse.ethz.ch