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

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.

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.

Differential nuclear expression of Yap in basal epithelial cells across the cornea and substrates of differing stiffness.

Corneal epithelium is maintained throughout life by well-orchestrated proliferation of limbal epithelial stem cells, followed by migration and maturation centripetally across the ocular surface. The present study sets out to explore the role tissue stiffness (compliance) may have in directing both differentiation and centripetal migration of limbal epithelial stem cells during homeostasis. For that, we analysed the localization of the Yes-associated protein (Yap), a transcriptional co-activator previously shown to mediate cellular response and mechanical stimuli. Using both models of ocular surface compliance and normal bovine corneas we evaluated the nuclear/cytoplasmic expression ratio of Yap. Expression levels within corneal epithelial cells were compared in situ between the limbus and central cornea, and in vitro between limbal epithelial stem cells expanded upon biomimetic collagen gels of increasing stiffness. Nuclear expression of Yap was shown to increase within the expanded cells upon substrates of increasing stiffness. Subsequently, Yap was used as a novel molecular probe to investigate the mechanical microenvironment within a normal ocular surface. The in situ localization of Yap was predominantly cytoplasmic within basal limbal epithelial cells and nuclear within basal central corneal epithelial cells. Furthermore, nuclear p63 expression was not co-localized with Yap in basal limbal epithelial cells. In conclusion, the current investigation provides new insights into the relationship between Yap and distinct cell populations across the ocular surface indicating that cells experience a different mechanical environment between the limbus and central cornea. A new hypothesis is put forward, in which centripetal differences in substrate stiffness drives the migration and differentiation of limbal epithelial stem cells, thus controlling corneal epithelium homeostasis.

Single-molecule force spectroscopy of G-protein-coupled receptors.

The applicability of single-molecule force spectroscopy (SMFS) to characterize membrane proteins in vitro is developing rapidly and opening a wide range of fascinating possibilities to study how intra- and intermolecular interactions determine their structural stability and functional state. In particular, understanding how molecular interactions contribute to the functional state of G-protein-coupled receptors (GPCRs) is of importance because they mediate most of our physiological responses and act as therapeutic targets for a broad spectrum of diseases. In our review we focus on SMFS to characterize GPCRs embedded in their physiologically relevant membranes and exposed to physiologically relevant conditions. SMFS uses a molecularly sharp stylus to grasp the terminal end of a GPCR and to quickly unfold the receptor while recording interaction forces. The positional accuracy of SMFS localizes these interactions to structural segments of the GPCR whereas the sensitivity of SMFS enables their stabilizing interaction forces to be quantified. To further investigate the kinetic, energetic and mechanical properties of the structural segments, dynamic SMFS (DFS) probes their stability over a wide range of loading rates. These parameters provide insight into the energy landscape that provides information on the structural and functional properties of the GPCRs. Selected highlights exemplify the application of SMFS to characterize inter- and intramolecular interactions, which change the properties of GPCRs in relation to their functional state (e.g., ligand binding), diseased state (e.g., mutation), or lipid environment such as cholesterol.

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.

Breakthrough instruments and products: DriveAFM for high-performance atomic force microscopy.

Atomic force microscopy is a powerful technique for measurement and mapping of nanoscale topography and electrical and mechanical sample properties. The Nanosurf DriveAFM is a new generation instrument that combines ease of use and high performance through full motorization, CleanDrive photothermal excitation, and a mechanical and electrical design that allows for both high-resolution and large-range imaging.

A stable disulfide-free gene-3-protein of phage fd generated by in vitro evolution.

Disulfide bonds provide major contributions to the conformational stability of proteins, and their cleavage often leads to unfolding. The gene-3-protein of the filamentous phage fd contains two disulfides in its N1 domain and one in its N2 domain, and these three disulfide bonds are essential for the stability of this protein. Here, we employed in vitro evolution to generate a disulfide-free variant of the N1-N2 protein with a high conformational stability. The gene-3-protein is essential for the phage infectivity, and we exploited this requirement for a proteolytic selection of stabilized protein variants from phage libraries. First, optimal replacements for individual disulfide bonds were identified in libraries, in which the corresponding cysteine codons were randomized. Then stabilizing amino acid replacements at non-cysteine positions were selected from libraries that were created by error-prone PCR. This stepwise procedure led to variants of N1-N2 that are devoid of all three disulfide bonds but stable and functional. The best variant without disulfide bonds showed a much higher conformational stability than the disulfide-containing wild-type form of the gene-3-protein. Despite the loss of all three disulfide bonds, the midpoints of the thermal transitions were increased from 48.5 degrees C to 67.0 degrees C for the N2 domain and from 60.0 degrees C to 78.7 degrees C for the N1 domain. The major loss in conformational stability caused by the removal of the disulfides was thus over-compensated by strongly improved non-covalent interactions. The stabilized variants were less infectious than the wild-type protein, probably because the domain mobility was reduced. Only a small fraction of the sequence space could be accessed by using libraries created by error-prone PCR, but still many strongly stabilized variants could be identified. This is encouraging and indicates that proteins can be stabilized by mutations in many different ways.

Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues.

Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein--MBP--and proteolipid protein--PLP--(respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5 kPa is the most suitable rigidity for oligodendrocyte differentiation.

pH-induced conformational change of the beta-barrel-forming protein OmpG reconstituted into native E. coli lipids.

A gating mechanism of the beta-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the beta-strands that form the transmembrane beta-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes.

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.

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.

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 approximately 6.5 degrees rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure.

Direct measurement of single-molecule visco-elasticity in atomic force microscope force-extension experiments.

Measuring the visco-elastic properties of biological macromolecules constitutes an important step towards the understanding of dynamic biological processes, such as cell adhesion, muscle function, or plant cell wall stability. Force spectroscopy techniques based on the atomic force microscope (AFM) are increasingly used to study the complex visco-elastic response of (bio-)molecules on a single-molecule level. These experiments either require that the AFM cantilever is actively oscillated or that the molecule is clamped at constant force to monitor thermal cantilever motion. Here we demonstrate that the visco-elasticity of single bio-molecules can readily be extracted from the Brownian cantilever motion during conventional force-extension measurements. It is shown that the characteristics of the cantilever determine the signal-to-noise (S/N) ratio and time resolution. Using a small cantilever, the visco-elastic properties of single dextran molecules were resolved with a time resolution of 8.3 ms. The presented approach can be directly applied to probe the dynamic response of complex bio-molecular systems or proteins in force-extension experiments.