Conformational dynamics of the alphaM3 transmembrane helix during acetylcholine receptor channel gating

Muscle acetylcholine receptors are synaptic ion channels that "gate" between closed- and open-channel conformations. We used Phi-value analysis to probe the transition state of the diliganded gating reaction with regard to residues in the M3, membrane-spanning helix of the muscle acetylcholine receptor alpha-subunit. Phi (a fraction between 1 and 0) parameterizes the extent to which a mutation changes the opening versus the closing rate constant and, for a linear reaction mechanism, the higher the Phi-value, the "earlier" the gating motion. In the upper half of alphaM3 the gating motions of all five tested residues were temporally correlated (Phi approximately 0.30) and serve to link structural changes occurring at the middle of the M2, pore-lining helix with those occurring at the interface of the extracellular and transmembrane domains. alphaM3 belongs to a complex and diverse set of synchronously moving parts that change structure relatively late in the channel-opening process. The propagation of the gating Brownian conformational cascade has a complex spatial distribution in the transmembrane domain.     

Tryptophan substitutions reveal the role of nicotinic acetylcholine receptor alpha-TM3 domain in channel gating: differences between Torpedo and muscle-type AChR

A recent tryptophan scanning of the alpha-TM3 domain of the Torpedo californica AChR demonstrated that this domain can modulate ion-channel gating [Guzman, G., Santiago, J., Ricardo, A., Martí-Arbona, R., Rojas, L., Lasalde-Dominicci, J. (2003) Biochemistry 42, 12243-12250]. Here we extend the study of the alpha-TM3 domain to the muscle-type AChR by examining functional consequences of single tryptophan substitutions at five conserved positions (alphaM282, alphaF284, alphaV285, alphaA287, and alphaI290) homologous to the alpha-TM3 positions that were recently characterized in the Torpedo AChR. Similarly to the Torpedo AChR, mutations alphaM282W and alphaV285W, which are presumed to face the interior of the protein, did not exhibit functional channel activity. Nevertheless, significant expression levels of these mutants were observed at the oocyte surface. In contrast to the Torpedo AChR, in the muscle-type AChR, tryptophan substitution at positions F284, A287, and I290 produces a significant increase in normalized macroscopic response. Single-channel recordings at low ACh concentration revealed that the increase in AChR sensitivity for the F284W, A287W, and I290W is due to an increase in the mean open duration. These results suggest that tryptophan substitution directly affects channel gating, primarily the channel closing rate. Our results suggest that residues facing the interior of the protein (i.e., alphaM282 and alphaV285) may similarly affect channel gating in Torpedo and muscle-type AChR. However, equivalent mutations (i.e., F284W and I290W) presumably facing the lipid environment display a very different functional response between these two AChR species.     

Tryptophan scanning mutagenesis in the alphaM3 transmembrane domain of the Torpedo californica acetylcholine receptor: functional and structural implications

The functional role of the alphaM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within alphaM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [(125)I]-alpha-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipid-exposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the alphaM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC(50). Taken together, our results suggest that alphaM3 contributes to the gating machinery of the nicotinic ACh receptor and that alphaM3 is comprised of a mixture of two types of helical structures.     

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the dM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the dM3 transmembrane domain were characterized by two-electrode voltage clamp and ¹²⁵I-labeled a-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300-301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid–exposed transmembrane domain and the nAChR gating machinery.     

Distinct structural elements in the first membrane-spanning segment of the epithelial sodium channel

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an alpha2betagamma or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within alphaM1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of alphaM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout alphaM1 (residues 110-130). We found that mutations within the amino-terminal part of alphaM1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of alphaM1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of alphaM1 is alpha-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of alphaM1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that alphaM1 is divided into two distinct regions.     

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the δM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the δM3 transmembrane domain were characterized by two-electrode voltage clamp and (125)I-labeled α-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300-301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery.     

Identification and function of conformational dynamics in the multidomain GTPase dynamin

Vesicle release upon endocytosis requires membrane fission, catalyzed by the large GTPase dynamin. Dynamin contains five domains that together orchestrate its mechanochemical activity. Hydrogen–deuterium exchange coupled with mass spectrometry revealed global nucleotide‐ and membrane‐binding‐dependent conformational changes, as well as the existence of an allosteric relay element in the α2^S helix of the dynamin stalk domain. As predicted from structural studies, FRET analyses detect large movements of the pleckstrin homology domain (PHD) from a ‘closed’ conformation docked near the stalk to an ‘open’ conformation able to interact with membranes. We engineered dynamin constructs locked in either the closed or open state by chemical cross‐linking or deletion mutagenesis and showed that PHD movements function as a conformational switch to regulate dynamin self‐assembly, membrane binding, and fission. This PHD conformational switch is impaired by a centronuclear myopathy‐causing disease mutation, S619L, highlighting the physiological significance of its role in regulating dynamin function. Together, these data provide new insight into coordinated conformational changes that regulate dynamin function and couple membrane binding, oligomerization, and GTPase activity during dynamin‐catalyzed membrane fission.     

Gating dynamics of the acetylcholine receptor extracellular domain

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the alpha-subunit of mouse muscle-type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a beta-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the beta-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the alpha-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Phi = 0.93) and is followed by the loop 2/loop 7 domain (Phi = 0.80). These movements precede that of the extracellular linker (Phi = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.     

Structural uncoupling between opposing domains of oxidized calmodulin underlies the enhanced binding affinity and inhibition of the plasma membrane Ca-ATPase

Stabilization of the plasma membrane Ca-ATPase (PMCA) in an inactive conformation upon oxidation of multiple methionines in the calcium regulatory protein calmodulin (CaM) is part of an adaptive cellular response to minimize ATP utilization and the generation of reactive oxygen species (ROS) under conditions of oxidative stress. To differentiate oxidant-induced structural changes that selectively modify the amino-terminal domain of CaM from those that modulate the conformational coupling between the opposing domains, we have engineered a tetracysteine binding motif within helix A in the amino-terminal domain of calmodulin (CaM) that permits the selective and rigid attachment of the conformationally sensitive fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)(2) (FlAsH-EDT(2)). The position of the FlAsH label in the amino-terminal domain provides a signal for monitoring its binding to the CaM-binding sequence of the PMCA. Following methionine oxidation, there is an enhanced binding affinity between the amino-terminal domain and the CaM-binding sequence of the PMCA. To identify oxidant-induced structural changes, we used frequency domain fluorescence anisotropy measurements to assess the structural coupling between helix A and the amino- and carboxyl-terminal domains of CaM. Helix A undergoes large amplitude motions in apo-CaM; following calcium activation, helix A is immobilized as part of a conformational switch that couples the opposing domains of CaM to stabilize the high-affinity binding cleft associated with target protein binding. Methionine oxidation disrupts the structural coupling between opposing globular domains of CaM, without affecting the calcium-dependent immobilization of helix A associated with activation of the amino-terminal domain to promote high-affinity binding to target proteins. We suggest that this selective disruption of the structural linkage between the opposing globular domains of CaM relieves steric constraints associated with high-affinity target binding, permitting the formation of new contact interactions between the amino-terminal domain and the CaM-binding sequence that stabilizes the PMCA in an inhibited conformation.     

Transient exposure of a buried phosphorylation site in an autoinhibited protein

Autoinhibition is a mechanism used to regulate protein function, often by making functional sites inaccessible through the interaction with a cis-acting inhibitory domain. Such autoinhibitory domains often display a substantial degree of structural disorder when unbound, and only become structured in the inhibited state. These conformational dynamics make it difficult to study the structural origin of regulation, including effects of regulatory post-translational modifications. Here, we study the autoinhibition of the Dbl Homology domain in the protein Vav1 by the so-called acidic inhibitory domain. We use molecular simulations to study the process by which a mostly unstructured inhibitory domain folds upon binding and how transient exposure of a key buried tyrosine residue makes it accessible for phosphorylation. We show that the inhibitory domain, which forms a helix in the bound and inhibited stated, samples helical structures already before binding and that binding occurs via a molten-globule-like intermediate state. Together, our results shed light on key interactions that enable the inhibitory domain to sample a finely tuned equilibrium between an inhibited and a kinase-accessible state.     

Toward elucidating the heat activation mechanism of the TRPV1 channel gating by molecular dynamics simulation

As a key cellular sensor, the TRPV1 cation channel undergoes a gating transition from a closed state to an open state in response to various physical and chemical stimuli including noxious heat. Despite years of study, the heat activation mechanism of TRPV1 gating remains enigmatic at the molecular level. Toward elucidating the structural and energetic basis of TRPV1 gating, we have performed molecular dynamics (MD) simulations (with cumulative simulation time of 3 μs), starting from the high‐resolution closed and open structures of TRPV1 solved by cryo‐electron microscopy. In the closed‐state simulations at 30°C, we observed a stably closed channel constricted at the lower gate (near residue I679), while the upper gate (near residues G643 and M644) is dynamic and undergoes flickery opening/closing. In the open‐state simulations at 60°C, we found higher conformational variation consistent with a large entropy increase required for the heat activation, and both the lower and upper gates are dynamic with transient opening/closing. Through ensemble‐based structural analyses of the closed state versus the open state, we revealed pronounced closed‐to‐open conformational changes involving the membrane proximal domain (MPD) linker, the outer pore, and the TRP helix, which are accompanied by breaking/forming of a network of closed/open‐state specific hydrogen bonds. By comparing the closed‐state simulations at 30°C and 60°C, we observed heat‐activated conformational changes in the MPD linker, the outer pore, and the TRP helix that resemble the closed‐to‐open conformational changes, along with partial formation of the open‐state specific hydrogen bonds. Some of the residues involved in the above key hydrogen bonds were validated by previous mutational studies. Taken together, our MD simulations have offered rich structural and dynamic details beyond the static structures of TRPV1, and promising targets for future mutagenesis and functional studies of the TRPV1 channel. Proteins 2016; 84:1938–1949. © 2016 Wiley Periodicals, Inc.     

Tryptophan scanning of the acetylcholine receptor's βM4 transmembrane domain: Decoding allosteric linkage at the lipid-protein interface with ion-channel gating

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that mediates fast excitatory synaptic transmission in the peripheral and central nervous systems. Changes in the structure and function of the AChR can lead to serious impairment of physiological processes. In this study, we combined site-directed mutagenesis, radioligand binding assays, electrophysiological recordings, and Fourier analyses to characterize the functional role and structural aspects of the βM4 transmembrane domain of the Torpedo AChR. We performed tryptophan replacements, from residues L438 through F455, along the βM4 transmembrane domain. Expression levels of mutants F439W-G450W and F452W-I454W produced peak currents similar to or lower than those in wild-type (WT). Tryptophan substitutions at positions L438 and T451 led to a deficiency in either subunit expression or receptor assembly. Mutations L440W, V442W, C447W, and S453W produced a gain-of-function response. Mutation F455W produced a loss of ion channel function. The periodicity profile of the normalized expression level (closed state) and EC50 (open state) revealed a minor conformational change of 0.4 residues/turn of the βM4 domain. These findings suggest that a minor movement of the βM4 domain occurs during channel activation.     

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.     

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane alpha-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 A at its narrowest, to 8.6 A at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.     

In vitro interaction between components of the inner membrane complex of the maltose ABC transporter of Escherichia coli : modulation by ATP

Interactions between domains of ATP-binding cassette (ABC) transporters are of great functional importance and yet are poorly understood. To gain further knowledge of these protein–protein interactions, we studied the inner membrane complex of the maltose transporter of Escherichia coli . We focused on interactions between the nucleotide-binding protein, MalK, and the transmembrane proteins, MalF and MalG. We incubated purified MalK with inverted membrane vesicles containing MalF and MalG. MalK bound specifically to MalF and MalG and reconstituted a functional complex. We used this approach and limited proteolysis with trypsin to show that binding and hydrolysis of ATP, inducing conformational changes in MalK, modulate its interaction with MalF and MalG. MalK in the reconstituted complex was less sensitive to protease added from the cytoplasmic side of the membrane, and one proteolytic cleavage site located in the middle of a putative helical domain of MalK was protected. These results suggest that the putative helical domain of the nucleotide-binding domains is involved, through its conformational changes, in the coupling between the transmembrane domains and ATP binding/hydrolysis at the nucleotide-binding domains.     

Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix α4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.     

Functional Analysis of the Transmembrane Domain in Paramyxovirus F Protein-Mediated Membrane Fusion

To enter cells, enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single-cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the paramyxovirus parainfluenza virus 5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion.     

Tilted, extended, and lying in wait: the membrane-bound topology of residues Lys-381-Ser-405 of the colicin E1 channel domain

The membrane-bound closed state (zero potential) of the helix 3 segment (Lys-381-Ser-405) of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane probe tethered to a single cysteine residue of each mutant protein. A number of fluorescence properties of the tethered bimane probe were measured for the soluble channel mutant proteins as well as for the membrane-bound proteins. A new method called helical periodicity surface analysis was employed to fit the fluorescence data to a harmonic wave function using four different statistical methods. The fit of the various data sets to a harmonic wave function indicated that the periodicity of helix 3 in the membrane-bound state is typical for an amphipathic alpha helix (3.7-4.0 residues per turn and an angular frequency between 90 and 97 degrees). Notably, upon membrane binding, helix 3 elongates from 15 residues (soluble structure) to 20 residues by a three- and two-residue extension at the N- and C-termini of the helix, respectively. Dual quencher analysis also revealed that helix 3 is appressed to the surface of the membrane with its N-terminus more deeply buried within the interfacial region of the bilayer than its C-terminus. Finally, contrary to a previous report, our data show that helices 3 and 4 remain separate and independent helices upon membrane association in the absence of a membrane potential.     

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane α-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12° relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 Å at its narrowest, to 8.6 Å at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.