Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex.

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b6f interaction is explained by FNR interaction with micelles of the undecyl ß-D maltoside (UDM) detergent micelles used to purify b6f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP+-binding domains of FNR caused by detergent. The enthalpy, ΔHo, and the entropy, ΔSo, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b6f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.

Novel bexarotene derivatives: Synthesis and cytotoxicity evaluation for glioma cells in 2D and 3D in vitro models.

Glioblastoma (GBM) is an aggressive and lethal form of brain cancer with a high invasion capacity and a lack of effective chemotherapeutics. Retinoid bexarotene (BXR) inhibits the neurospheroidal colony formation and migration of primary glioblastoma cells but has side effects. To enhance the BXR glioblastoma selectivity and cytotoxicity, we chemically modified it at the carboxyl group with either nitroethanolamine (NEA) bearing a NO-donating group (a well-known bioactivity enhancer; BXR-NEA) or with a dopamine (DA) moiety (to represent the highly toxic for various tumor cells N-acyldopamine family; BXR-DA). These two novel compounds were tested in the 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Both BXR-DA and BXR-NEA were found to be more toxic for rat C6 and human U-87MG glioma cells than the initial BXR. After 24 h incubation of the cells (monolayer culture) with the drugs, the IC50 values were in the range of 28-42, and 122-152 µM for BXR derivatives and BXR, respectively. The cell death occurred via apoptosis according to the annexin staining and caspase activation. The tumor spheroids demonstrated higher resistance to the treatment compared to that one of the monolayer cultures. BXR-DA and BXR-NEA were more specific against tumor cells than the parental drug, in particular the selectivity index was 1.8-2.7 vs. 1.3-1.5, respectively. Moreover, they inhibited cell migration more effectively than parental BXR according to a scratch assay. Cell spreading from the tumor spheroids was also inhibited. Thus, the obtained BXR derivatives could be promising for glioblastoma treatment.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System.

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 µM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki = 16.7 ± 1.5 µM and αKi = 51.4 ± 4.1 µM for AChE and Ki = 70.5 ± 6.3 µM and αKi = 214 ± 17 µM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

An evolutionarily conserved iron-sulfur cluster underlies redox sensory function of the Chloroplast Sensor Kinase.

Photosynthetic efficiency depends on equal light energy conversion by two spectrally distinct, serially-connected photosystems. The redox state of the plastoquinone pool, located between the two photosystems, is a key regulatory signal that initiates acclimatory changes in the relative abundance of photosystems. The Chloroplast Sensor Kinase (CSK) links the plastoquinone redox signal with photosystem gene expression but the mechanism by which it monitors the plastoquinone redox state is unclear. Here we show that the purified Arabidopsis and Phaeodactylum CSK and the cyanobacterial CSK homologue, Histidine kinase 2 (Hik2), are iron-sulfur proteins. The Fe-S cluster of CSK is further revealed to be a high potential redox-responsive [3Fe-4S] center. CSK responds to redox agents with reduced plastoquinone suppressing its autokinase activity. Redox changes within the CSK iron-sulfur cluster translate into conformational changes in the protein fold. These results provide key insights into redox signal perception and propagation by the CSK-based chloroplast two-component system.

Structural insight into the role of the Ton complex in energy transduction.

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.

Trans-membrane Signaling in Photosynthetic State Transitions: REDOX- AND STRUCTURE-DEPENDENT INTERACTION IN VITRO BETWEEN STT7 KINASE AND THE CYTOCHROME b6f COMPLEX.

Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the cytochrome b6f complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the cytochrome b6f complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the b6f complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the b6f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b6f complex by quinol oxidation.

The Colicin E1 TolC-Binding Conformer: Pillar or Pore Function of TolC in Colicin Import?

The mechanism by which the drug export protein TolC is utilized for import of the cytotoxin colicin E1 across the outer membrane and periplasmic space is addressed. Studies of the initial binding of colicin E1 with TolC, occlusion of membrane-incorporated TolC ion channels, and the structure underlying the colicin-TolC complex were based on the interactions with TolC of individual colicin translocation domain (T-domain) peptides from a set of 19 that span different segments of the T-domain. These studies led to identification of a short 20-residue segment 101-120, a "TolC box", located near the center of the colicin T-domain, which is necessary for binding of colicin to TolC. Omission of this segment eliminated the ability of the T-domain to occlude TolC channels and to co-elute with TolC on a size-exclusion column. Far-ultraviolet circular dichroism spectral and thermal stability analysis of the structure of T-domain peptides implies (i) a helical hairpin conformation of the T-domain, (ii) the overlap of the TolC-binding site with a hinge of the helical hairpin, and (iii) a TolC-dependent stage of colicin import in which a central segment of the T-domain in a helical hairpin conformation binds to the TolC entry port following initial binding to the BtuB receptor. These studies provide the first structure-based information about the interaction of colicin E1 with the unique TolC protein. The model inferred for binding of the T-domain to TolC implies reservations about the traditional model for colicin import in which TolC functions to provide a channel for translocation of the colicin in an unfolded state across the bacterial outer membrane and a large part of the periplasmic space.

Structure-function of cyanobacterial outer-membrane protein, Slr1270: homolog of Escherichia coli drug export/colicin import protein, TolC.

Compared to thylakoid and inner membrane proteins in cyanobacteria, no structure-function information is available presently for integral outer-membrane proteins (OMPs). The Slr1270 protein from the cyanobacterium Synechocystis 6803, over-expressed in Escherichia coli, was refolded, and characterized for molecular size, secondary structure, and ion-channel function. Refolded Slr1270 displays a single band in native-electrophoresis, has an α-helical content of 50-60%, as in E. coli TolC with which it has significant secondary-structure similarity, and an ion-channel function with a single-channel conductance of 80-200pS, and a monovalent ion (K(+):Cl(-)) selectivity of 4.7:1. The pH-dependence of channel conductance implies a role for carboxylate residues in channel gating, analogous to that in TolC.

A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex.

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation-reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme-heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation-reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure-function parameter of membrane protein complexes.

Self-oscillating Water Chemiluminescence Modes and Reactive Oxygen Species Generation Induced by Laser Irradiation; Effect of the Exclusion Zone Created by Nafion.

Samples of water inside and outside an exclusion zone (EZ), created by Nafion swollen in water, were irradiated at the wavelength l = 1264 nm, which stimulates the electronic transition of dissolved oxygen from the triplet state to the excited singlet state. This irradiation induces, after a long latent period, chemiluminescence self-oscillations in the visible and near UV spectral range, which last many hours. It occurs that this effect is EZ-specific: the chemiluminescence intensity is twice lower than that from the bulk water, while the latent period is longer for the EZ. Laser irradiation causes accumulation of H2O2, which is also EZ-specific: its concentration inside the EZ is less than that in the bulk water. These phenomena can be interpreted in terms of a model of decreasing O2 content in the EZ due to increased chemical activity of bisulfite anions (HSO3-), arisen as the result of dissociation of terminal sulfonate groups of the Nafion. The wavelet transform analysis of the chemiluminescence intensity from the EZ and the bulk water gives, that self-oscillations regimes occurring in the liquid after the latent period are the determinate processes. It occurred that the chemiluminescence dynamics in case of EZ is characterized by a single-frequency self-oscillating regime, whereas in case of the bulk water, the self-oscillation spectrum consists of three spectral bands.

Methods for studying interactions of detergents and lipids with α-helical and ß-barrel integral membrane proteins.

Methods for studying interactions of protein with lipids and detergents are described for representatives of two major classes of membrane proteins: (1) the α-helical hetero-oligomeric integral cytochrome b6 f complex of oxygenic photosynthesis from cyanobacteria, and (2) the outer membrane ß-barrel proteins BtuB and OmpF from Gram-negative Escherichia coli bacteria. Details are presented on the use of detergents for purification and crystallization of the b6 f complex as well as a method for lipid exchange. The positions of detergent and lipid molecules, which define eight potential lipid-binding sites in the b6 f complex, are described. Differences in detergent strategies for isolation and crystallization of ß-barrel proteins relative to those for oligomeric helical membrane proteins are discussed, and purification and assessment of protein quality by circular dichroism (CD) is presented.

Pathways of colicin import: utilization of BtuB, OmpF porin and the TolC drug-export protein.

Pathway I. Group A nuclease colicins parasitize and bind tightly (Kd ≤ 10(-9) M) to the vitamin B12 receptor on which they diffuse laterally in the OM (outer membrane) and use their long (≥100 Å; 1 Å=0.1 nm) receptor-binding domain as a 'fishing pole' to locate the OmpF porin channel for translocation. Crystal structures of OmpF imply that a disordered N-terminal segment of the colicin T-domain initiates insertion. Pathway II. Colicin N does not possess a 'fishing pole' receptor-binding domain. Instead, it uses OmpF as the Omp (outer membrane protein) for reception and translocation, processes in which LPS (lipopolysaccharide) may also serve. Keio collection experiments defined the LPS core that is used. Pathway III. Colicin E1 utilizes the drug-export protein TolC for import. CD spectra and thermal-melting analysis predict: (i) N-terminal translocation (T) and central receptor (BtuB) -binding (R) domains are predominantly α-helical; and (ii) helical coiled-coil conformation of the R-domain is similar to that of colicins E3 and Ia. Recombinant colicin peptides spanning the N-terminal translocation domain defined TolC-binding site(s). The N-terminal 40-residue segment lacks the ordered secondary structure. Peptide 41-190 is helical (78%), co-elutes with TolC and occluded TolC channels. Driven by a trans-negative potential, peptides 82-140 and 141-190 occluded TolC channels. The use of TolC for colicin E1 import implies that the interaction of this colicin with the other Tol proteins does not occur in the periplasmic space, but rather through Tol domains in the cytoplasmic membrane, thus explaining colicin E1 cytotoxicity towards a strain in which a 234 residue periplasmic TolA segment is deleted.

Oxygen-dependent auto-oscillations of water luminescence triggered by the 1264 nm radiation.

A 5-min exposure of air-saturated bidistilled water to low-intensity laser infrared radiation at the wavelength of the electronic transition of dissolved oxygen to the singlet state ((3)∑(g)(-)→ (1)Δ(g)) induces, after a long latent period, auto-oscillations of water luminescence in the blue-green region, which last many hours. Laser irradiation causes the accumulation of hydrogen peroxide, which depends on the concentration of dissolved oxygen. The auto-oscillations do not arise if water is irradiated beyond the oxygen absorption band and if the oxygen is removed from water. The wavelet transform analysis of luminescence records indicates that there are two characteristic periods of pulsations of about 300 and 1150 s. The results obtained suggest that auto-oscillations are triggered by photoinduced singlet oxygen (1)Δ(g), and this phenomenon is closely related to formation of hydrogen peroxide.

Mobility of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex.

Diffusion of two Escherichia coli outer membrane proteins-the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages-was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient (D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05±0.01 µm2/s and 0.10±0.02 µm2/s, respectively, over a timescale of 25-150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27±0.06 µm2/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006±0.002 µm2/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.

Primary events in the colicin translocon: FRET analysis of colicin unfolding initiated by binding to BtuB and OmpF.

Cellular import of colicin E3 is initiated by high affinity binding of the colicin receptor-binding (R) domain to the vitamin B(12) (BtuB) receptor in the Escherichia coli outer membrane. The BtuB binding site, at the apex of its extended coiled-coil R-domain, is distant from the C-terminal nuclease domain that must be imported for expression of cytotoxicity. Based on genetic analysis and previously determined crystal structures of the R-domain bound to BtuB, and of an N-terminal disordered segment of the translocation (T) domain inserted into the OmpF porin, a translocon model for colicin import has been inferred. Implicit in the model is the requirement for unfolding of the colicin segments inserted into OmpF. FRET analysis was employed to study colicin unfolding upon interaction with BtuB and OmpF. A novel method of Cys-specific dual labeling of a native polypeptide, which allows precise placement of donor and acceptor fluorescent dyes on the same polypeptide chain, was developed. A decrease in FRET efficiency between the translocation and cytotoxic domains of the colicin E3 was observed upon colicin binding in vitro to BtuB or OmpF. The two events were independent and additive. The colicin interactions with BtuB and OmpF have a major electrostatic component. The R-domain Arg399 is responsible for electrostatic interaction with BtuB. It is concluded that free energy for colicin unfolding is provided by binding of the R- domain to BtuB and binding/insertion of the T-domain to/into OmpF.

Crystal structures of the OmpF porin: function in a colicin translocon.

The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 A OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co-crystallization of OmpF with the unfolded 83 residue glycine-rich N-terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 A structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly-glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno-extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.

Helical alpha-synuclein forms highly conductive ion channels.

Alpha-synuclein (alphaS) is a cytosolic protein involved in the etiology of Parkinson's disease (PD). Disordered in an aqueous environment, alphaS develops a highly helical conformation when bound to membranes having a negatively charged surface and a large curvature. It exhibits a membrane-permeabilizing activity that has been attributed to oligomeric protofibrillar forms. In this study, monomeric wild-type alphaS and two mutants associated with familial PD, E46K and A53T, formed ion channels with well-defined conductance states in membranes containing 25-50% anionic lipid and 50% phosphatidylethanolamine (PE) in the presence of a trans-negative potential. Another familial mutant, A30P, known to have a lower membrane affinity, did not form ion channels. Ca2+ prevented channel formation when added to membranes before alphaS and decreased channel conductance when added to preformed channels. In contrast to the monomer, membrane permeabilization by oligomeric alphaS was not characterized by formation of discrete channels, a requirement for PE lipid, or a membrane potential. Channel activity, alpha-helical content, thermal stability of membrane-bound alphaS determined by far-UV CD, and lateral mobility of alphaS bound to planar membranes measured by fluorescence correlation spectroscopy were correlated. It was inferred that discrete ion channels with well-defined conductance states were formed in the presence of a membrane potential by one or several molecules of monomeric alphaS in an alpha-helical conformation and that such channels may have a role in the normal function and/or pathophysiology of the protein.

Structure of the complex of the colicin E2 R-domain and its BtuB receptor. The outer membrane colicin translocon.

The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-A crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg(452) at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.

The colicin E3 outer membrane translocon: immunity protein release allows interaction of the cytotoxic domain with OmpF porin.

The crystal structure previously obtained for the complex of BtuB and the receptor binding domain of colicin E3 forms a basis for further analysis of the mechanism of colicin import through the bacterial outer membrane. Together with genetic analysis and studies on colicin occlusion of OmpF channels, this implied a colicin translocon consisting of BtuB and OmpF that would transfer the C-terminal cytotoxic domain (C96) of colicin E3 through the Escherichia coli outer membrane. This model does not, however, explain how the colicin attains the unfolded conformation necessary for transfer. Such a conformation change would require removal of the immunity (Imm) protein, which is bound tightly in a complex with the folded colicin E3. In the present study, it was possible to obtain reversible removal of Imm in vitro in a single column chromatography step without colicin denaturation. This resulted in a mostly unordered secondary structure of the cytotoxic domain and a large decrease in stability, which was also found in the receptor binding domain. These structure changes were documented by near- and far-UV circular dichroism and intrinsic tryptophan fluorescence. Reconstitution of Imm in a complex with C96 or colicin E3 restored the native structure. C96 depleted of Imm, in contrast to the native complex with Imm, efficiently occluded OmpF channels, implying that the presence of tightly bound Imm prevents its unfolding and utilization of the OmpF porin for subsequent import of the cytotoxic domain.

Lipid dependence of the channel properties of a colicin E1-lipid toroidal pore.

Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 A, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.