Activation energy for pore opening in lipid membranes under an electric field.

The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-µm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.

Pore-forming toxins as tools for polymer analytics: From sizing to sequencing.

We report here on the nanopore resistive pulse sensing (Np-RPS) method, involving pore-forming toxins as tools for polymer analytics at single molecule level. Np-RPS is an electrical method for the label-free detection of single molecules. A molecule interacting with the pore causes a change of the electrical resistance of the pore, called a resistive pulse, associated with a measurable transient current blockade. The features of the blockades, in particular their depth and duration, contain information on the molecular properties of the analyte. We first revisit the history of Np-RPS, then we discuss the effect of the configuration of the molecule/nanopore interaction on the molecular information that can be extracted from the signal, illustrated in two different regimes that either favor molecular sequencing or molecular sizing. Specifically, we focus on the sizing regime and on the use of two different pore-forming toxins, staphylococcal α-hemolysin (αHL) and aerolysin (AeL) nanopores, for the characterization of water-soluble polymers (poly-(ethylene glycol), (PEG)), homopeptides, and heteropeptides. We discuss how nanopore sizing of polymers could be envisioned as a new approach for peptide/protein sequencing.

Electrophysiology on Channel-Forming Proteins in Artificial Lipid Bilayers: Next-Generation Instrumentation for Multiple Recordings in Parallel.

Artificial lipid bilayers have been used for several decades to study channel-forming pores and ion channels in membranes. Until recently, the classical two-chamber setups have been primarily used for studying the biophysical properties of pore forming proteins. Within the last 10 years, instruments for automated lipid bilayer measurements have been developed and are now commercially available. This chapter focuses on protein purification and reconstitution of channel-forming proteins into lipid bilayers using a classic setup and on the commercially available systems, the Orbit mini and Orbit 16.

A Conserved Proline Hinge Mediates Helix Dynamics and Activation of Rhodopsin.

Despite high-resolution crystal structures of both inactive and active G protein-coupled receptors (GPCRs), it is still not known how ligands trigger the large structural change on the intracellular side of the receptor since the conformational changes that occur within the extracellular ligand-binding region upon activation are subtle. Here, we use solid-state NMR and Fourier transform infrared spectroscopy on rhodopsin to show that Trp2656.48 within the CWxP motif on transmembrane helix H6 constrains a proline hinge in the inactive state, suggesting that activation results in unraveling of the H6 backbone within this motif, a local change in dynamics that allows helix H6 to swing outward. Notably, Tyr3017.48 within activation switch 2 appears to mimic the negative allosteric sodium ion found in other family A GPCRs, a finding that is broadly relevant to the mechanism of receptor activation.

Activity of the Gramicidin A Ion Channel in a Lipid Membrane with Switchable Physical Properties.

Lipid bilayer membranes formed from the artificial 1,3-diamidophospholipid Pad-PC-Pad have the remarkable property that their hydrophobic thickness can be modified in situ: the particular arrangement of the fatty acid chains in Pad-PC-Pad allows them to fully interdigitate below 37 °C, substantially thinning the membrane with respect to the noninterdigitated state. Two stimuli, traversing the main phase transition temperature of the lipid or addition of cholesterol, have previously been shown to disable the interdigitated state. Both manipulations cause an increase in hydrophobic thickness of about 25 Å due to enhanced conformational entropy of the lipids. Here, we characterize the interdigitated state using electrophysiological recordings from free-standing lipid-membranes formed on micro structured electrode cavity arrays. Compared to standard membranes made from 1,2-diphytanoyl-sn-glycero-3-phosphocholin (DPhPC), pure Pad-PC-Pad membranes at room temperature had lowered electroporation threshold and higher capacitance. Ion channel formation by the peptide Gramicidin A was clearly facilitated in pure Pad-PC-Pad membranes at room temperature, with activity occurring at significantly lower peptide concentrations and channel dwell times increased by 2 orders of magnitude with respect to DPhPC-membranes. Both elevation of temperature beyond the phase transition and addition of cholesterol reduced channel dwell times, as expected if the reduced membrane thickness stabilized channel formation due to decreased hydrophobic mismatch.

The Multifaceted Antibacterial Mechanisms of the Pioneering Peptide Antibiotics Tyrocidine and Gramicidin S.

Cyclic ß-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent.IMPORTANCE Cyclic ß-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of ß-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them.

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR.

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.

Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation.

The 11-cis retinal chromophore is tightly packed within the interior of the visual receptor rhodopsin and isomerizes to the all-trans configuration following absorption of light. The mechanism by which this isomerization event drives the outward rotation of transmembrane helix H6, a hallmark of activated G protein-coupled receptors, is not well established. To address this question, we use solid-state NMR and FTIR spectroscopy to define the orientation and interactions of the retinal chromophore in the active metarhodopsin II intermediate. Here we show that isomerization of the 11-cis retinal chromophore generates strong steric interactions between its ß-ionone ring and transmembrane helices H5 and H6, while deprotonation of its protonated Schiff's base triggers the rearrangement of the hydrogen-bonding network involving residues on H6 and within the second extracellular loop. We integrate these observations with previous structural and functional studies to propose a two-stage mechanism for rhodopsin activation.

Automated formation of lipid membrane microarrays for ionic single-molecule sensing with protein nanopores.

Efficient use of membrane protein nanopores in ionic single-molecule sensing requires technology for the reliable formation of suspended molecular membranes densely arrayed in formats that allow high-resolution electrical recording. Here, automated formation of bimolecular lipid layers is shown using a simple process where a poly(tetrafluoroethylene)-coated magnetic bar is remotely actuated to perform a turning motion, thereby spreading phospholipid in organic solvent on a nonpolar surface containing a <1 mm(2) 4 × 4 array of apertures with embedded microelectrodes (microelectrode cavity array). Parallel and high-resolution single-molecule detection by single nanopores is demonstrated on the resulting bilayer arrays, which are shown to form by a classical but very rapid self-assembly process. The technique provides a robust and scalable solution for the problem of reliable, automated formation of multiple independent lipid bilayers in a dense microarray format, while preserving the favorable electrical properties of the microelectrode cavity array.

Highly conserved tyrosine stabilizes the active state of rhodopsin.

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.

SEIRA spectroscopy on a membrane receptor monolayer using lipoprotein particles as carriers.

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.

Tracking G-protein-coupled receptor activation using genetically encoded infrared probes.

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.

Sequential rearrangement of interhelical networks upon rhodopsin activation in membranes: the Meta II(a) conformational substate.

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.

Structural impact of the E113Q counterion mutation on the activation and deactivation pathways of the G protein-coupled receptor rhodopsin.

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.

Monitoring of actin unfolding by room temperature tryptophan phosphorescence.

Slow intramolecular mobility of native and inactivated actin from rabbit skeletal muscle during the process of protein unfolding induced by GdnHCl was studied using tryptophan room temperature phosphorescence (RTP). By this method, the conclusion was confirmed that an essentially unfolded intermediate preceded the formation of inactivated actin [Turoverov et al. Biochemistry (2002) 41, 1014-1019]. It was found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting the high lability of its structure. Symbate changes of integral intensity and the mean lifetime of RTP during the U* --> I transition suggests a gradual increase of the number of monomers incorporated in the associate (U* --> I(1)... --> I(n)... --> I(15)), which is accompanied by an increase of structural rigidity. The rate of inactivated actin formation (I identical with I(15)) is shown to increase with the increase of protein concentration. It is shown that, no matter what the means of inactivation, actin transition to the inactivated state is accompanied by a significant increase of both integral intensity and the mean lifetime of RTP, suggesting that inactivated actin has a rigid structure.