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Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C19F3-labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19F-19F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

Disruption of an interhelical salt bridge between the retinal protonated Schiff base linked to H7 and Glu113 on H3 is one of the decisive steps during activation of rhodopsin. Using previously established stabilization strategies, we engineered a stabilized E113Q counterion mutant that converted rhodopsin to a UV-absorbing photoreceptor with deprotonated Schiff base and allowed reconstitution into native-like lipid membranes. Fourier-transform infrared difference spectroscopy reveals a deprotonated Schiff base in the photoproducts of the mutant up to the active state Meta II, the absence of the classical pH-dependent Meta I/Meta II conformational equilibrium in favor of Meta II, and an anticipation of active state features under conditions that stabilize inactive photoproduct states in wildtype rhodopsin. Glu181 on extracellular loop 2, is found to be unable to maintain a counterion function to the Schiff base on the activation pathway of rhodopsin in the absence of the primary counterion, Glu113. The Schiff base becomes protonated in the transition to Meta III. This protonation is, however, not associated with a deactivation of the receptor, in contrast to wildtype rhodopsin. Glu181 is suggested to be the counterion in the Meta III state of the mutant and appears to be capable of stabilizing a protonated Schiff base in Meta III, but not of constraining the receptor in an inactive conformation.

Cell-free expression is a rapidly developing tool for the efficient production of membrane proteins with enormous potential. Here we report on the design of a setup for parallel high-resolution electrophysiological recordings allowing for the screening of functional activity of ion channels synthesized in-vitro in various lipid environments.The developed recording chamber is based on a SU-8 coated printed circuit board containing 4 cavities (50µm) with individual microelectrodes (Micro-Electrode-Cavity-Array (MECA) as well as a common ground electrode. 4 suspended lipid bilayers can be self-assembled on the nonpolar chip surface from phospholipids in organic solvent. The MECA-PCB is connected to a multichannel amplifier capable of simultaneous recording of electrical activity from the ion channels functionally reconstituted into the bilayers with high-bandwidth and low noise (

Oxidation of membrane lipids is a ubiquitous phenomenon in eukaryotic cells occurring as a consequence of the generation of reactive oxygen species by mitochondrial or peroxisomal activity and irradiation with photons or ions. Lipid hydroperoxidation, the primary step of the oxidative reactions, has been shown to induce molecular area increase, enhanced fluctuations and tube formation in giant unilamellar vesicles. Surprisingly, however, despite a 75% loss of elastic modulus, even fully hydroperoxidized membranes can remain effective barriers against small molecule diffusion1, contrary to membranes including other downstream oxidation products2,3,4. We were therefore interested to ascertain whether membrane permeability to ions of pure lipid bilayers is affected by lipid hydroperoxidation. Using a chip-based device for automated bilayer formation and parallel recording from suspended synthetic lipid bilayers (Orbit-16, Nanion), we compared responses to positive and negative voltages in the range of 50 to 200 mV of POPC- and 100% hydroperoxidized POPC (POPC-OOH)1 membranes formed on 50 µm-diam. microelectrode cavities of the MECA16 chip (Ionera). While control POPC membranes showed a stable conductance in the range of a few pS without evidence for pore-formation, POPC-OOH membranes responded with fluctuating conductance increases already at 50 mV. Conductance grew in a voltage and time-dependent manner, reaching values of several nS at +/-150 mV. Remarkably, the voltage induced conductance was stable over time and did not recover during a 1 s waiting period between pulses, showing cumulative behavior from one pulse to the next. Conductance fluctuations were characterized by multiple preferred levels separated by several tens of pS without evident equidistant periodicity. Taken together, these findings suggest that in response to voltage, hydroperoxidized POPC membranes form multiple, stable voltage-dependent, ion-permeant pores with conductances similar to protein ion channels.(1) Weber. et al. Lipid Oxidation Induces Structural Changes in Biomimetic Membranes. Soft Matter 2014, 10, 4241-4247.(2) Caetano et al. Photo-induced Destruction of Giant Vesicles in Methylene Blue Solutions. Langmuir, 2007, 23, 130-137.(3) Mertins et al. Physical Damage on Giant Vesicles Membrane as a Result of Methylene Blue Photoirradiation, Biophys. J. 2014, 106, 162-171.(4) Runas, K. A.; Malmstadt, N. Low Levels of Lipid Oxidation Radically Increase the Passive Permeability of Lipid Bilayers. Soft Matter 2015, 11, 499-505.

Light-induced isomerization of the 11- cis- retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg135 3.50 and Met257 6.40 forms in Meta II, consistent with the outward rotation of H6 and breaking of the dark-state Glu134 3.49 -Arg135 3.50 -Glu247 6.30 ionic lock. We also show that Tyr223 5.58 and Tyr306 7.53 form molecular contacts with Met257 6.40 . Together these results reveal that the crystal structure of opsin in the region of the ionic lock reflects the active state of the receptor. We further demonstrate that Tyr223 5.58 and Ala132 3.47 in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr223 5.58 to phenylalanine or mutation of Ala132 3.47 to leucine decreases the lifetime of the Meta II intermediate. Furthermore, the Y223F mutation is coupled to structural changes in EL2. In contrast, mutation of Tyr306 7.53 to phenylalanine shows only a moderate influence on the Meta II lifetime and is not coupled to EL2.

Photon absorption by rhodopsin is proposed to lead to an activation pathway that is described by the extended reaction scheme Meta I==Meta II(a)==Meta II(b)==Meta II(b)H(+), where Meta II(b)H(+) is thought to be the conformational substate that activates the G protein transducin. Here we test this extended scheme for rhodopsin in a membrane bilayer environment by investigating lipid perturbation of the activation mechanism. We found that symmetric membrane lipids having two unsaturated acyl chains, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), selectively stabilize the Meta II(a) substate in the above mechanism. By combining FTIR and UV-visible difference spectroscopy, we characterized the structural and functional changes involved in the transition to the Meta II(a) intermediate, which links the inactive Meta I intermediate with the Meta II(b) states formed by helix rearrangement. Besides the opening of the Schiff base ionic lock, the Meta II(a) substate is characterized by an activation switch in a conserved water-mediated hydrogen-bonded network involving transmembrane helices H1/H2/H7, which is sensed by its key residue Asp83. On the other hand, movement of retinal toward H5 and its interaction with another interhelical H3/H5 network mediated by His211 and Glu122 is absent in Meta II(a). The latter rearrangement takes place only in the subsequent transition to Meta II(b), which has been previously associated with movement of H6. Our results imply that activating structural changes in the H1/H2/H7 network are triggered by disruption of the Schiff base salt bridge and occur prior to other chromophore-induced changes in the H3/H5 network and the outward tilt of H6 in the activation process.

Recent studies using pore forming membrane proteins in single-molecule detection have concentrated exclusively on analyte interaction with the so-called “canonic pore”, displaying a certain conductance under given ionic conditions and presumably corresponding to a pore structure assembled from a particu-lar number of monomers. For instance, for the pore-forming toxin alpha-hemolysin, a conductance of approximately 1 nS in 1 M KCl at pH 7.5 is thought to correspond to a heptameric assembly, while a smaller conductance level is likely to be due to pores formed by a smaller number of monomers [1-4] Using a recently developed microarray device for parallel and high-resolution recording from suspended micro-bilayres (Microelectrode cavity array, MECA [5-7] we have begun to study the properties of the lower con-ductance form of aHL-mediated pores. The adavntage of the parallel recording system for these studies is that single canonical pores can be recorded simultaneously with low conductance forms under identical conditions. We found that low-conductance variants tend to appear after prolonged incubation of mono-mers in recording solution (3 M KCl). Interestingly, these smaller pores are blocked by poly-ethyleneglycol (PEG) oligomers in a fashion very similar to the larger canonical pores [8], showing a simi-lar resolution of PEG mass [6,9-11]. However, surprisingly, the relative position of the maxima in the histo-gram of relative residual conductances are shifted to larger values for the smaller pores. This finding has potential implications for the mechanism of the block by PEG, in that it suggests that PEG entry into the pore adds a resistance in series with the resistance of the internal constriction site.

Proteins able to form “donut shaped” assemblies have been expressed in Escherichia coli and used for reversible in vitro assembly of cylindrical yoctowells (yoctolitre: 1 yL = 10−24 L). Their assembly and disassembly were studied spectroscopically and fibres ranging from molecular to multibundle structures have been assembled.

Surface-enhanced infrared absorption (SEIRA) difference spectroscopy can probe reactions in a protein monolayer tethered to a nanostructured gold surface. SEIRA studies of membrane proteins, however, remain challenging due to sample stability, effects of the metal surface on function, and the need for a membrane-mimicking environment. Here we demonstrate and characterize a model system for membrane receptor investigations using SEIRA spectroscopy. The system employs nanoscale apolipoprotein bound bilayer (NABB) particles, similar to discoidal high-density lipoprotein particles, as soluble carriers for the G-protein-coupled receptor rhodopsin. The His-tag of the engineered apolipoprotein allows for selective binding of the NABBs to a Ni-NTA modified surface, while the lipid environment of the particle ensures stability and protection of the embedded receptor. Using SEIRA spectroscopy, we followed specific binding of rhodopsin-loaded NABB particles to the surface and formation of a membrane protein monolayer. Functionality of the photoreceptor in the immobilized NABBs was probed by SEIRA difference spectroscopy confirming protein conformational changes associated with photoactivation. Orientation of the immobilized NABB particles was assessed by comparing SEIRA data with polarized attenuated total reflection-Fourier-transform infrared spectroscopy. Thus, SEIRA difference spectroscopy supported by the NABB technology provides a promising approach for further functional studies of transmembrane receptors.

High-throughput electrophysiological recordings in artificial membranes have a great potential for pharmaceutical screening and nanopore analytics. In addition to parallelization and automation of lipid bilayers formation, different protein reconstitution protocols are required depending on the concrete application.We report on functional reconstitutions of three classes of pore-forming proteins into suspended lipid bilayers on Micro Electrode Cavity Array (MECA). Three reconstitution strategies have been employed and optimized depending on the protein structure: self-insertion for pore-forming toxins, transfer from detergent micelles for porins and vesicle fusion for ion channels.By optimizing protein concentration and facilitating spontaneous insertion with short voltage pulses just below the electroporation threshold, a controlled insertion of alpha-hemolysin was achieved. Exactly one pore per bilayer was inserted in over 50% of bilayers in the array, which is substantially higher than the 37% predicted by the Poisson distribution.We investigated and optimized insertion of outer membrane porins OmpF, MspA and OccK1 into bilayer arrays via detergent dilution. Best strategy was to use mild nonionic detergents and sequentially dilute protein stock solution to minimize influence of the detergent on the membrane. Protein injection in the immediate vicinity of a bilayer array and thorough mixing of the solution were decisive factors for homogeneous distribution of functional porins.Reconstitution via detergent micelles was found to be problematic for ion channels, but proteovesicle fusion was successful. The tetrameric potassium channel KcsA was expressed in vitro with cotranslational insertion into lipid vesicles or nanodiscs. Transfer from nanodiscs into suspended bilayers was shown to be inefficient. Additionally, the presence of nanodiscs substantially destabilized the membranes. For proteovesicles best fusion rates were achieved upon addition of negatively charged phospholipids followed by extrusion of the recovered proteovesicles through a polycarbonate filter.