Conformational changes of colicin Ia channel-forming domain upon membrane binding: a solid-state NMR study

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected (13)C and (15)N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val C(alpha) signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the alpha-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by (2)H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding.     

Solid-state NMR investigation of the dynamics of the soluble and membrane-bound colicin Ia channel-forming domain.

Solid-state NMR spectroscopy was employed to study the molecular dynamics of the colicin Ia channel domain in the soluble and membrane-bound states. In the soluble state, the protein executes small-amplitude librations (with root-mean-square angular fluctuations of 0-10 degrees ) in the backbone and larger-amplitude motions (16-17 degrees ) in the side chains. Upon membrane binding, the motional amplitudes increase significantly for both the backbone (12-16 degrees ) and side chains (23-29 degrees ), as manifested by the reduction in the C-H and H-H dipolar couplings and (15)N chemical shift anisotropy. These motions occur not only on the pico- to nanosecond time scales, but also on the microsecond time scale, as revealed by the (1)H rotating-frame spin-lattice relaxation times. Average motional correlation times of 0.8 and 1.2 micros were extracted for the soluble and membrane-bound states, respectively. In comparison, both forms of the colicin Ia channel domain are completely immobile on the millisecond scale. These results indicate that the colicin Ia channel domain has enhanced conformational mobility in the lipid bilayer compared to the soluble state. This membrane-induced mobility increase is consistent with the loss of tertiary structure of the protein in the membrane, which was previously suggested by the extended helical array model [Zakharov et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4282-4287]. An extended structure would also facilitate protein interactions with the mobile lipids and thus increase the protein internal motions. We speculate that the large mobility of the membrane-bound colicin Ia channel domain is a prerequisite for channel opening in the presence of a voltage gradient.     

Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End?

Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an approximately 68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.     

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.     

The colicin Ia receptor,Cir, is also the translocator for colicin Ia

Colicin Ia, a channel‐forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill Escherichia coli, it must cross this membrane. The crystal structure of Ia receptor‐binding domain bound to Cir, a 22‐stranded plugged β‐barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no ‘second receptor’ has ever been identified for TonB‐dependent colicins, such as Ia. We show that if the receptor‐binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild‐type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N‐terminal translocation (T) domain and its channel‐forming C‐terminal domain. Deletion of colicin Ia's receptor‐binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia's receptor‐binding domain, Cir also binds weakly to its translocation domain.     

Oligomeric Structure of Colicin Ia Channel in Lipid Bilayer Membranes

Colicin Ia is a soluble, harpoon-shaped bacteriocin which translocates across the periplasmic space of sensitive Escherichia coli cell by parasitizing an outer membrane receptor and forms voltage-gated ion channels in the inner membrane. This process leads to cell death, which has been thought to be caused by a single colicin Ia molecule. To directly visualize the three-dimensional structure of the channel, we generated two-dimensional crystals of colicin Ia inserted in lipid-bilayer membranes and determined a ∼17 three-dimensional model by electron crystallography. Supported by velocity sedimentation, chemical cross-linking and single-particle image analysis, the three-dimensional structure is a crown-shaped oligomer enclosing a ∼35 Å-wide extrabilayer vestibule. Our study suggests that lipid insertion instigates a global conformational change in colicin Ia and that more than one molecule participates in the channel architecture with the vestibule, possibly facilitating the known large scale peptide translocation upon channel opening.Colicin Ia is a pore-forming water-soluble bacterial toxin produced by some strains of Escherichia coli to kill other competing bacteria (1, 2). It belongs to a functionally and structurally similar group of proteins that also includes colicins A (3), E1 (4), and N (5). Each of these proteins consist of three domains with distinct properties; the receptor domain (R), which binds a specific outer membrane receptor on the target cell, and the translocation domain (T) at the N terminus, responsible for traversing the outer membrane and the periplasmic space to deliver the channel-forming domain (C) at the C terminus to the bacterial inner membrane. The bundle of 10 α-helices that compose the C domain changes its conformation to form a voltage-gated ion channel in the plasma membrane. Opening of the channel produces an efflux of ions that depletes the cellular energy resources and ultimately leads to cell death.The x-ray structure of full-length, soluble colicin Ia (69 kDa) has been determined (6). The monomeric molecule is mostly α-helical, with the R domain separated from the T and C domains by a pair of unusually long (∼160 Å) α-helices thought possibly to span the periplasmic space during channel formation (6). The C domain is characterized by two hydrophobic helices (VIII and IX; residues Ala-580—Ile-612) that is surrounded by the remaining eight largely amphipathic α-helices. The same structural motif for the C domain is conserved in other members of the colicin family and is also present in the channel-forming domains of diphtheria toxin, exotoxin A, and the Bcl family of pro- and anti-apoptotic proteins (7). This pair of helices, termed the hydrophobic hairpin, is instrumental in driving the initial membrane insertion event (8) that is followed by a series of large scale pH and voltage-dependent conformational changes in the C domain, resulting in the opening of the ion channel in the plasma membrane (9, 10). In the absence of a high resolution membrane-inserted structure of a channel-forming colicin, solid-state NMR (11, 12), streptavidin binding (8) and cross-linking of site-directed cysteine mutants (9) have suggested that the initial membrane-bound intermediate exists as a two-dimensional helical array of the eight amphipathic helices (I-VII and X) spread across the membrane surface, with the hydrophobic helices (VIII and IX) embedded in the bilayer. A recent electron paramagnetic resonance study using preparations of spin-labeled ColA proteoliposomes has supported a similar umbrella model where the eight amphipathic helices reside at the air-water interface for the closed-channel state (13). Biotin-labeled cysteine mutants have also been used to determine how much of the C domain (aside from the hydrophobic hairpin) crosses the plasma membrane (14, 15) for colicin Ia. A large region of the amphipathic sequence (helices II-V; residues Leu-474—Tyr-541) has been found to cross from the cis to the trans side of the membrane in planar lipid bilayer experiments, resulting in a four-transmembrane segment molecule that is thought to form the ion channel.Because the 12–13 residue α-helices of the C domain are well short of the ∼20 residues required to span the plasma membrane, it has been proposed that conformational changes causing helix extension take place during the channel formation process. ¹³C spin diffusion NMR has indicated that whereas the overall secondary structure of the C domain is preserved, most of the helices undergo “opening,” and modulation of the tertiary structure allows for the required extension of the helices to cross the plasma membrane and form the channel (16). The internal structure of the colicin Ia channel has been investigated by examining the effect of different nonelectrolyte molecules on the single-channel conductance in planar lipid bilayer membranes (17). It was determined that the diameter at the cis entrance (equivalent to the outside of the cell) is 18 Å, and the diameter at the trans entrance (inside the membrane) is 10 Å, with a 7 Å diameter constriction located in close proximity to the trans entrance of the channel. More recent studies (18) employing the substituted cysteine accessibility method to determine what residues line the open colicin Ia channel suggest an hourglass-shaped pore with the most constricted part near the cis rather than the trans side, as opposed to the conclusion of Krasilnikov et al. (17). Both studies point to a pore constriction inside the membrane, and as pointed out by Kienker et al. 18), there exist plausible explanations to reconcile some of the differing results. The large diameter of the colicin Ia channel coupled with the studies which indicate that each colicin Ia molecule contributes four transmembrane segments in the membrane integrated state (14) suggests that the ion channel is formed by a multimer of colicin Ia molecules. However, all of the past studies directed at determining the oligomeric state of any of the colicin channels indicate a monomeric structure. The question as to how a four-transmembrane monomeric protein can form an ion channel of sufficient diameter to allow the passage of ions as large as tetraethyl ammonium (19) has remained unanswered.In this work we have subjected colicin Ia incorporated into lipid bilayer membranes to structural and biochemical investigations. We show, based on cross-linking and velocity sedimentation experiments, single-particle analysis of electron micrographs and results from electron crystallographic analysis of two-dimensional crystals of colicin Ia that the protein forms oligomers upon insertion into the bilayer. The suggested architecture of this oligomer based on the ∼17 Å resolution three-dimensional model and the biological implications, are discussed.     

Unfolding pathway of the colicin E1 channel protein on a membrane surface

The channel-forming domain of colicin E1 is composed of a soluble helical bundle which, upon membrane binding, unfolds to form an extended, two-dimensional helical net in the membrane interfacial layer. To characterize the pathway of unfolding of the protein and the structure of the surface-bound intermediate, the time-course of intra-protein distance changes and unfolding on a millisecond time-scale were determined from the kinetics of changes in the efficiency of fluorescence resonance energy transfer, and of the donor-acceptor overlap integral, between each of six individual tryptophan residues and a Cys-conjugated energy transfer acceptor (C509-AEDANS). Comparison of the rate constants revealed the following order of events associated with unfolding of the protein at the membrane surface: (A) movement of the hydrophobic core helices VIII-IX, coincident with a small change in Trp-Cys509 distances of the outer helices; (B) unfolding of surface helices in the helical bundle in the order: helix I, helices III, IV, VI, VII, and helix V; (C) a slow (time-scale, seconds) condensation of the surface-bound helices. The rate of protein unfolding events increased with increasing anionic lipid content. Unfolding did not occur below the lipid thermal phase transition, indicating that unfolding requires mobility in the interfacial layer. The structure of the two-dimensional membrane-bound intermediate in the steady-state was inferred to consist of a quasi-circular arrangement of eight helices embedded in the membrane interfacial layer and anchored by the hydrophobic helical hairpin. The pathway of unfolding of the colicin channel at the membrane surface, catalyzed by electrostatic and hydrophobic forces, is the first described for a membrane-active protein. It is proposed that the pathway and principles described for the colicin protein are relevant to membrane protein import.     

Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes

The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells. The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation. The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed. Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes. Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate. The nature of the open-channel structure is discussed.     

Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes

The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells. The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation. The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed. Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes. Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate. The nature of the open-channel structure is discussed.     

Effects of anionic lipid and ion concentrations on the topology and segmental mobility of colicin Ia channel domain from solid-state NMR

Channel-forming colicins are bacterial toxins that spontaneously insert into the inner cell membrane of sensitive bacteria to form voltage-gated ion channels. It has been shown that the channel current and the conformational flexibility of colicin E1 channel domain depend on the membrane surface potential, which is regulated by the anionic lipid content and the ion concentration. To better understand the dependence of colicin structure and dynamics on the membrane surface potential, we have used solid-state NMR to investigate the topology and segmental motion of the closed state of colicin Ia channel-forming domain in membranes of different anionic lipid contents and ion concentrations. Colicin Ia channel domain was reconstituted into membranes with different POPG and KCl concentrations. 1H spin diffusion experiments indicate that the protein contains a small domain that inserts into the hydrophobic center of the 70% anionic membrane, similar to when it binds to the 25% anionic membrane. Measurements of C-H and N-H dipolar couplings indicate that, on the sub-microsecond time scale, the protein has the least segmental mobility under the high-salt and low-anionic lipid condition, which has the most physiological membrane surface potential. Measurement of millisecond time scale motions yielded similar results. These suggest that optimal channel activity requires the protein to have sufficient segmental rigidity so that entire helices can undergo cooperative conformational motions that are required for translocating the channel-forming helices across the lipid bilayer upon voltage activation.     

On the role of lipid in colicin pore formation

Insights into the protein-membrane interactions by which the C-terminal pore-forming domain of colicins inserts into membranes and forms voltage-gated channels, and the nature of the colicin channel, are provided by data on: (i) the flexible helix-elongated state of the colicin pore-forming domain in the fluid anionic membrane interfacial layer, the optimum anionic surface charge for channel formation, and voltage-gated translocation of charged regions of the colicin domain across the membrane; (ii) structure-function data on the voltage-gated K(+) channel showing translocation of an arginine-rich helical segment through the membrane; (iii) toroidal channels formed by small peptides that involve local participation of anionic lipids in an inverted phase. It is proposed that translocation of the colicin across the membrane occurs through minimization of the Born charging energy for translocation of positively charged basic residues across the lipid bilayer by neutralization with anionic lipid head groups. The resulting pore structure may consist of somewhat short, ca. 16 residues, trans-membrane helices, in a locally thinned membrane, together with surface elements of inverted phase lipid micelles.     

NMR determination of protein partitioning into membrane domains with different curvatures and application to the influenza M2 peptide

The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use ³¹P and ¹³C solid-state NMR to examine M2-induced membrane curvature. M2(22–46), which includes only the transmembrane (TM) helix, and M2(21–61), which contains an additional amphipathic helix, are studied. ³¹P chemical shift lineshapes indicate that M2(21–61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22–46) has minimal effect. The lamellar and isotropic domains have distinct ³¹P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. ³¹P- and ¹³C-detected ¹H T₂ relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21–61) preferentially binds to the high-curvature domain. ³¹P linewidths indicate that the isotropic vesicles induced by M2(21–61) are 10–35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides.     

Histidine 440 controls the opening of colicin E1 channels in a lipid-dependent manner

The in vitro activity of many pore-forming toxins, in particular, the rate of increase in the membrane conductance induced by the channel-forming domain (P178) of colicin E1 is maximum at an acidic pH. However, after P178 binding at acidic conditions, a subsequent pH shift from 4 to 6 on both sides of the planar bilayer lipid membrane caused a large increase in the trans-membrane current which was solely due to an increase in the number of open channels. This effect required the presence of anionic lipid. Replacing the His440 residue of P178 by alanine eliminated the pH-shift effect thereby showing that it is associated with deprotonation of this histidine residue. It was concluded that alkalinization-induced weakening of the electrostatic interactions between colicin and the membrane surface facilitates conformational changes required for the transition of membrane-bound colicin molecules to an active channel state.     

Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia. Received: 12 November 1996/Revised: 23 January 1997     

Scanning the membrane-bound conformation of helix 1 in the colicin E1 channel domain by site-directed fluorescence labeling

Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membrane-bound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic alpha-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic alpha-helix (3.7 +/- 0.1 residues per turn and 97 +/- 3.0 degrees, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin.     

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.     

Major transmembrane movement associated with colicin Ia channel gating

Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltage-dependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine residues introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin- containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a disulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution). The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist.     

Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10

The pre-channel state of helices 6, 7, and 10 (Val⁴⁴⁷–Gly⁴⁷⁵ and Ile⁵⁰⁸–Ile⁵²²) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain.     

Backbone resonance assignments of the C2 domain of coagulation factor VIII

Factor VIII (FVIII, other clotting factors are named similarly) is a glycoprotein that circulates in the plasma bound to von Willebrand factor. During the blood coagulation cascade, activated FVIII (FVIIIa) binds to FIXa and activates FX in the presence of calcium ions and phospholipid membranes. The C1 and C2 domains mediate membrane binding that is essential for activation of the FVIIIa–FIXa complex. Here, ¹H, ¹³C, and ¹⁵N backbone chemical shift assignments are reported for the C2 domain of FVIII, including assignments for the residues in solvent-exposed loops. The NMR resonance assignments, along with further structural studies of membrane-bound FVIII, will advance understanding of blood-clotting protein interactions.     

Membrane topology of the colicin E1 channel using genetically encoded fluorescence

The membrane topology of the colicin E1 channel domain was studied by fluorescence resonance energy transfer (FRET). The FRET involved a genetically encoded fluorescent amino acid (coumarin) as the donor and a selectively labeled cysteine residue tethered with DABMI (4-(dimethylamino)phenylazophenyl-4'-maleimide) as the FRET acceptor. The fluorescent coumarin residue was incorporated into the protein via an orthogonal tRNA/aminoacyl-tRNA synthetase pair that allowed selective incorporation into any site within the colicin channel domain. Each variant harbored a stop (TAG) mutation for coumarin incorporation and a cysteine (TGT) mutation for DABMI attachment. Six interhelical distances within helices 1-6 were determined using FRET analysis for both the soluble and membrane-bound states. The FRET data showed large changes in the interhelical distances among helices 3-6 upon membrane association providing new insight into the membrane-bound structure of the channel domain. In general, the coumarin-DABMI FRET interhelical efficiencies decreased upon membrane binding, building upon the umbrella model for the colicin channel. A tentative model for the closed state of the channel domain was developed based on current and previously published FRET data. The model suggests circular arrangement of helices 1-7 in a clockwise direction from the extracellular side and membrane interfacial association of helices 1, 6, 7, and 10 around the central transmembrane hairpin formed by helices 8 and 9.