Christian A. Bippes

High-resolution atomic force microscopy and spectroscopy of native membrane proteins

Membranes confining cells and cellular compartments are essential for life. Membrane proteins are molecular machines that equip cell membranes with highly sophisticated functionality. Examples of such functions are signaling, ion pumping, energy conversion, molecular transport, specific ligand binding, cell adhesion and protein trafficking. However, it is not well understood how most membrane proteins work and how the living cell regulates their function. We review how atomic force microscopy (AFM) can be applied for structural and functional investigations of native membrane proteins. High-resolution time-lapse AFM imaging records membrane proteins at work, their oligomeric state and their dynamic assembly. The AFM stylus resembles a multifunctional toolbox that allows the measurement of several chemical and physical parameters at the nanoscale. In the single-molecule force spectroscopy (SMFS) mode, AFM quantifies and localizes interactions in membrane proteins that stabilize their folding and modulate their functional state. Dynamic SMFS discloses fascinating insights into the free energy landscape of membrane proteins. Single-cell force spectroscopy quantifies the interactions of live cells with their environment to single-receptor resolution. In the future, technological progress in AFM-based approaches will enable us to study the physical nature of biological interactions in more detail and decipher how cells control basic processes.

Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

Gap junction channels regulate cell-cell communication by passing metabolites, ions, and signaling molecules. Gap junction channel closure in cells by acidification is well documented; however, it is unknown whether acidification affects connexins or modulating proteins or compounds that in turn act on connexins. Protonated aminosulfonates directly inhibit connexin channel activity in an isoform-specific manner as shown in previously published studies. High-resolution atomic force microscopy of force-dissected connexin26 gap junctions revealed that in HEPES buffer, the pore was closed at pH < 6.5 and opened reversibly by increasing the pH to 7.6. This pH effect was not observed in non-aminosulfonate buffers. Increasing the protonated HEPES concentration did not close the pore, indicating that a saturation of the binding sites occurs at 10 mm HEPES. Analysis of the extracellular surface topographs reveals that the pore diameter increases gradually with pH. The outer connexon diameter remains unchanged, and there is a ∼6.5° rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure.

pH-Dependent Interactions Guide the Folding and Gate the Transmembrane Pore of the β-Barrel Membrane Protein OmpG

The physical interactions that switch the functional state of membrane proteins are poorly understood. Previously, the pH-gating conformations of the beta-barrel forming outer membrane protein G (OmpG) from Escherichia coli have been solved. When the pH changes from neutral to acidic the flexible extracellular loop L6 folds into and closes the OmpG pore. Here, we used single-molecule force spectroscopy to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At acidic pH, we detected a pH-dependent interaction at loop L6. This interaction changed the (un)folding of loop L6 and of beta-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pH-dependent interactions change the folding of a peptide loop to gate the transmembrane pore. They further demonstrate how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore.

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.

Competing interactions stabilize pro- and anti-aggregant conformations of human Tau.

Aggregation of Tau into amyloid-like fibrils is a key process in neurodegenerative diseases such as Alzheimer. To understand how natively disordered Tau stabilizes conformations that favor pathological aggregation, we applied single-molecule force spectroscopy. Intramolecular interactions that fold polypeptide stretches of ∼19 and ∼42 amino acids in the functionally important repeat domain of full-length human Tau (hTau40) support aggregation. In contrast, the unstructured N terminus randomly folds long polypeptide stretches >100 amino acids that prevent aggregation. The pro-aggregant mutant hTau40ΔK280 observed in frontotemporal dementia favored the folding of short polypeptide stretches and suppressed the folding of long ones. This trend was reversed in the anti-aggregant mutant hTau40ΔK280/PP. The aggregation inducer heparin introduced strong interactions in hTau40 and hTau40ΔK280 that stabilized aggregation-prone conformations. We show that the conformation and aggregation of Tau are regulated through a complex balance of different intra- and intermolecular interactions.

High-throughput single-molecule force spectroscopy for membrane proteins

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ∼400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ∼200 (AdiC) and ∼400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

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

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

Digital force-feedback for protein unfolding experiments using atomic force microscopy

Since its invention in the 1990s single-molecule force spectroscopy has been increasingly applied to study protein (un-)folding, cell adhesion, and ligand–receptor interactions. In most force spectroscopy studies, the cantilever of an atomic force microscope (AFM) is separated from a surface at a constant velocity, thus applying an increasing force to folded bio-molecules or bio-molecular bonds. Recently, Fernandez and co-workers introduced the so-called force-clamp technique. Single proteins were subjected to a defined constant force allowing their life times and life time distributions to be directly measured. Up to now, the force-clamping was performed by analogue PID controllers, which require complex additional hardware and might make it difficult to combine the force-feedback with other modes such as constant velocity. These points may be limiting the applicability and versatility of this technique. Here we present a simple, fast, and all-digital (software-based) PID controller that yields response times of a few milliseconds in combination with a commercial AFM. We demonstrate the performance of our feedback loop by force-clamp unfolding of single Ig27 domains of titin and the membrane proteins bacteriorhodopsin (BR) and the sodium/proton antiporter NhaA.

Single-Molecule Force Spectroscopy of Membrane Proteins from Membranes Freely Spanning Across Nanoscopic Pores

Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.

Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

Significance Proton-dependent oligopeptide transporters are attractive candidates for drug research. To understand their functional modulation by drugs, we applied single-molecule force spectroscopy and characterized how peptide transport facilitated by the dipeptide and tripeptide permease A (DtpA) from Escherichia coli is inhibited. In the unbound state DtpA embedded in the physiologically relevant membrane adopts two alternate conformations, which differ mainly in whether the transmembrane α-helix TMH2 is stabilized. TMH2 contains residues that are important for ligand binding and substrate affinity. Inhibitor (Lys[Z-NO2]-Val) binding to DtpA significantly strengthens the interactions stabilizing TMH2 and guides DtpA to populate the inhibited conformation. Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di- and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO2]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward- and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO2]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.