Models of voltage-dependent conformational changes in NaChBac channels

Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an α-helix, and the last part forms a 3₁₀ helix, whereas in the closed model, the first part of S4 forms a 3₁₀ helix, and the last part forms an α-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the α-helical part of S4 and a simple axial translation for the 3₁₀ portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and K_v channels are discussed.     

Peptidic HIV integrase inhibitors derived from HIV gene products: Structure–activity relationship studies

Structure–activity relationship studies were conducted on HIV integrase (IN) inhibitory peptides which were found by the screening of an overlapping peptide library derived from HIV-1 gene products. Since these peptides located in the second helix of Vpr are considered to have an α-helical conformation, Glu-Lys pairs were introduced into the i and i + 4 positions to increase the helicity of the lead compound possessing an octa-arginyl group. Ala-scan was also performed on the lead compound for the identification of the amino acid residues responsible for the inhibitory activity. The results indicated the importance of an α-helical structure for the expression of inhibitory activity, and presented a binding model of integrase and the lead compound.     

Effect of side chain length on intrahelical interactions between carboxylate- and guanidinium-containing amino acids

The charge-containing hydrophilic functionalities of encoded charged amino acids are linked to the backbone via different numbers of hydrophobic methylenes, despite the apparent electrostatic nature of protein ion pairing interactions. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on ion pairing interactions, α-helical peptides containing Zbb–Xaa (i, i + 3), (i, i + 4) and (i, i + 5) (Zbb = carboxylate-containing residues Aad, Glu, Asp in decreasing length; Xaa = guanidinium residues Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by circular dichroism spectroscopy (CD). The helicity of Aad- and Glu-containing peptides was similar and mostly pH independent, whereas the helicity of Asp-containing peptides was mostly pH dependent. Furthermore, the Arg-containing peptides consistently exhibited higher helicity compared to the corresponding Agp-, Agb-, and Agh-containing peptides. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb–Xaa (i, i + 3) and (i, i + 4) interactions mainly involved the χ ₁ dihedral combinations (g+, g+) and (g?, g+), respectively. These low energy conformations were also observed in intrahelical Asp–Arg and Glu–Arg salt bridges of natural proteins. Accordingly, Asp and Glu provides variation in helix characteristics associated with Arg, but Aad does not provide features beyond those already delivered by Glu. Importantly, nature may have chosen the side chain length of Arg to support helical conformations through inherent high helix propensity coupled with stabilizing intrahelical ion pairing interactions with the carboxylate-containing residues.     

Role of hydrophobic and ionic forces in the movement of S4 of the Shaker potassium channel

Abstract

Voltage-gated ion (K⁺, Na⁺, Ca²⁺) channels contain a pore domain (PD) surrounded by four voltage sensing domains (VSD). Each VSD is made up of four transmembrane helices, S1–S4. S4 contains 6–7 positively charged residues (arginine/lysine) separated two hydrophobic residues, whereas S1–S3 contribute to two negatively charged clusters. These structures are conserved among all members of the voltage-gated ion channel family and play essential roles in voltage gating. The role of S4 charged residues in voltage gating is well established: During depolarization, they move out of the membrane electric field, exerting a mechanical force on channel gates, causing them to open. However, the role of the intervening hydrophobic residues in voltage sensing is unclear. Here we studied the role of these residues in the prototypical Shaker potassium channel. We have altered the physicochemical properties of both charged and hydrophobic positions of S4 and examined the effect of these modifications on the gating properties of the channel. For this, we have introduced cysteines at each of these positions, expressed the mutants in Xenopus oocytes, and examined the effect of in situ addition of charge, via Cd²⁺, on channel gating by two-electrode voltage clamp. Our results reveal a face of the S4 helix (comprising residues L358, L361, R365 and R368) where introduction of charge at hydrophobic positions destabilises the closed state and removal of charges from charged positions has an opposite effect. We propose that hydrophobic residues play a crucial role in limiting gating to a physiological voltage range.     

Helix-helix interactions and their impact on protein motifs and assemblies

Protein secondary structure elements are arranged in distinct structural motifs such as four-α-helix bundle, 8α/8β TIM-barrel, Rossmann dinucleotide binding fold, assembly of a helical rod. Each structural motif is characterized by a particular type of helix-helix interactions. A unique pattern of contacts is formed by interacting helices of the structural motif. In each type of fold, edges of the helix surface, which participate in the formation of helix-helix contacts with preceding and following helices, differ. This work shows that circular arrangements of the four, eight, and sixteen α-helices, which are found in the four-α-helical motif, TIM-barrel 8α/8β fold, and helical rod of 16.3¯ helices per turn correspondingly, can be associated with the mutual positioning of the edges of the helix surfaces. Edges (i, i+1)−(i+1, i+2) of the helix surface are central for the interhelical contacts in a four-α-helix bundle. Edges (i, i+1)−(i+2, i+3) are involved in the assembly of four-α-helix subunits into helical rod of a tobacco mosaic virus and a three-helix fragment of a Rossmann fold. In 8α/8β TIM-barrel fold, edges (i, i+1)−(i+5, i+6) are involved in the octagon arrangement. Approximation of a cross section of each motif with a polygon (n-gon, n=4, 8, 16) shows that a good correlation exists between polygon interior angles and angles formed by the edges of helix surfaces.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Accessibility of Four Arginine Residues on the S4 Segment of the Bacillus halodurans Sodium Channel

The voltage-gated Na⁺ channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1–S6), with a pore-forming region composed of segments S5 and S6 and a voltage-sensing domain composed of segments S1–S4. The S4 segment forms the core of the voltage sensor. We explored the accessibility of four arginine residues on the S4 segment of NaChBac, which are positioned at every third position from each other. These arginine residues on the S4 segment were replaced with cysteines using site-directed mutagenesis. Na⁺ currents were recorded using the whole-cell configuration of the patch-clamp technique. We tested the effect of the sulfhydryl reagents applied from inside and outside the cellular space in the open and closed conformations. Structural models of the voltage sensor of NaChBac were constructed based on the recently crystallized KvAP and Kv1.2 K⁺ channels to visualize arginine residue accessibility. Our results suggest that arginine accessibility did not change significantly between the open and closed conformations, supporting the idea of a small movement of the S4 segment during gating. Molecular modeling of the closed conformation also supported a small movement of S4, which is mainly characterized by a rotation and a tilt along the periphery of the pore. Interestingly, the second arginine residue of the S4 segment (R114) was accessible to sulfhydryl reagents from both sides of the membrane in the closed conformation and, based on our model, seemed to be at the junction of the intracellular and extracellular water crevices.     

NMR structures of the histidine-rich peptide LAH4 in micellar environments: membrane insertion, pH-dependent mode of antimicrobial action, and DNA transfection

The LAH4 family of histidine-rich peptides exhibits potent antimicrobial and DNA transfection activities, both of which require interactions with cellular membranes. The bilayer association of the peptides has been shown to be strongly pH-dependent, with in-planar alignments under acidic conditions and transmembrane orientations when the histidines are discharged. Therefore, we investigated the pH- and temperature-dependent conformations of LAH4 in DPC micellar solutions and in a TFE/PBS solvent mixture. In the presence of detergent and at pH 4.1, LAH4 adopts helical conformations between residues 9 and 24 concomitantly with a high hydrophobic moment. At pH 6.1, a helix-loop-helix structure forms with a hinge encompassing residues His¹⁰-Ala¹³. The data suggest that the high density of histidine residues and the resulting electrostatic repulsion lead to both a decrease in the pK values of the histidines and a less stable α-helical conformation of this region. The hinged structure at pH 6.1 facilitates membrane anchoring and insertion. At pH 7.8, the histidines are uncharged and an extended helical conformation including residues 4-21 is again obtained. LAH4 thus exhibits a high degree of conformational plasticity. The structures provide a stroboscopic view of the conformational changes that occur during membrane insertion, and are discussed in the context of antimicrobial activity and DNA transfection.     

Conformation of Vespa basalis mastoparan-B in trifluoroethanol-containing aqueous solution

Mastoparan-B, a tetradecapeptide isolated from the venom of the hornet Vespa basalis, belongs to the mastoparan analogs of vespid venom with the lysine residues common for all mastoparan family toxins at positions 4, 11 and 12. Here we use ¹H-NMR spectroscopy and hybrid distance geometry-simulated annealing calculation to investigate its three-dimensional structure in trifluoroethanol-containing aqueous solution. The calculated structure shows that residues 3–14 adopt an amphiphilic α-helical structure in which the residues with hydrophilic side chains (i.e. Lys-4, Ser-5, Ser-8, Lys-11, Lys-12) are located on one side and the residues with hydrophobic side chains (i.e. Leu-3, Ile-6, Trp-9, A a-10, Val-13, Leu-14) located on the other side of the molecule. The overall structural features are very similar to the conformation of mastoparan-X reconstituted in vesicles [Wakamatsu et al. (1992) Biochemistry 31, 5654–5660] in spite of the substitutions made for eight residues with distinctly different hydrophobicity. These substitutions lead to a larger hydrophobic moment for the α-helical segment and further mobilized N-terminal. This study will help reveal the conformational significance of mastoparan toxins with respect to their potency and activity in G protein regulation.     

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.     

Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

'In dopaminergic neurons, α-synuclein (αS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of αS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free state and the lipid-bound αS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s^− 1. Partitioning of the binding modes is dependent on lipid/αS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of αS adopts an α-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this “dark state.” Titration of αS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by αS.     

Substitution of a hydrophobic residue alters the conformational stability of Shaker K+ channels during gating and assembly.

A leucine residue at position 370 (L370) in 29-4 Shaker K+ channels resides within two overlapping sequence motifs conserved among most voltage-gated channels: the S4 segment and a leucine heptad repeat. Here we investigate the effects observed upon substitution of L370 with many other uncharged amino acid residues. We find that smaller or more hydrophilic residues produce greater alterations in both activation and inactivation gating than does substitution with other large hydrophobic residues. In addition, subunits containing less conservative substitutions at position 370 are restricted in their assembly with wild-type subunits and are unlikely to form homomultimeric channel complexes. Consistent with the idea that L370 influences the tertiary structure of these channels, the results indicate that L370 undergoes specific hydrophobic interactions during the conformational transitions of gating; similar interactions may take place during the folding, insertion, or assembly of Shaker K+ channel subunits.     

Ca 2+-induced self-assembly in designed peptides with optimally spaced gamma-carboxyglutamic acid residues

We have previously elucidated a new paradigm for the metal ion-induced helix-helix assembly in the natural γ-carboxyglutamic acid (Gla)-containing class of conantokin (con) peptides, typified by con-G and a variant of con-T, con-T[K7Gla], independent of the hydrophobic effect. In these “metallo-zipper” structures, Gla residues spaced at i, i + 4, i + 7, i + 11 intervals, which is similar to the arrangement of a and d residues in typical heptads of coiled-coils, coordinate with Ca²⁺ and form specific antiparallel helical dimers. In order to evaluate the common role of Gla residues in peptide self-assembly, we extend herein the same Gla arrangement to designed peptides: NH₂-(γLSγEAK)₃-CONH₂ (peptide 1) and NH₂-γLSγEAKγLSγQANγLSγKAE-CONH₂ (peptide 2). Peptide 1 and peptide 2 exhibit no helicity alone, but undergo structural transitions to helical conformations in the presence of a variety of divalent cations. Sedimentation equilibrium ultracentrifugation analyses showed that peptide 1 and peptide 2 form helical dimers in the presence of Ca²⁺, but not Mg²⁺. Folding and thiol-disulfide rearrangement assays with Cys-containing peptide variants indicated that the helical dimers are mixtures of antiparallel and parallel dimers, which is different from the strict antiparallel strand orientations of con-G and con-T[K7γGla] dimers. These findings suggest that the Gla arrangement, i, i + 4, i + 7, i + 11, i + 14, plays a key role in helix formation, without a strict adherence to strand orientation of the helical dimer.     

Alanine Scanning of the S6 Segment Reveals a Unique and cAMP-sensitive Association between the Pore and Voltage-dependent Opening in HCN Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels resemble Shaker K⁺ channels in structure and function. In both, changes in membrane voltage produce directionally similar movement of positively charged residues in the voltage sensor to alter the pore structure at the intracellular side and gate ion flow. However, HCNs open when hyperpolarized, whereas Shaker opens when depolarized. Thus, electromechanical coupling between the voltage sensor and gate is opposite. A key determinant of this coupling is the intrinsic stability of the pore. In Shaker, an alanine/valine scan of residues across the pore, by single point mutation, showed that most mutations made the channel easier to open and steepened the response of the channel to changes in voltage. Because most mutations likely destabilize protein packing, the Shaker pore is most stable when closed, and the voltage sensor works to open it. In HCN channels, the pore energetics and vector of work by the voltage sensor are unknown. Accordingly, we performed a 22-residue alanine/valine scan of the distal pore of the HCN2 isoform and show that the effects of mutations on channel opening and on the steepness of the response of the channel to voltage are mixed and smaller than those in Shaker. These data imply that the stabilities of the open and closed pore are similar, the voltage sensor must apply force to close the pore, and the interactions between the pore and voltage sensor are weak. Moreover, cAMP binding to the channel heightens the effects of the mutations, indicating stronger interactions between the pore and voltage sensor, and tips the energetic balance toward a more stable open state.Hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channels are similar in structure and function to Shaker K⁺ channels (1–3). As in Shaker, HCN channels are comprised of four subunits, which each consist of six predicted membrane-spanning segments (S1–S6). The S1–S4 segments form the voltage-sensing domain, and the S5 and S6 segments, the pore-forming domain. The S4 segment in both channels contains positive charges that move similarly in response to changes in membrane voltage (4–6), to then alter the pore structure at the intracellular side of the S6 segment; this region functions as a voltage-controlled gate to cation flow (7–10). Despite these similarities, HCN channels are opened by hyperpolarization of the membrane potential, whereas Shaker channels open in response to depolarization. Thus, the electromechanical coupling between the voltage sensor and the gate is reversed in these two channels.A key determinant of this coupling is the intrinsic stability of the closed and open conformations of the pore. In Shaker channels, it has been proposed that the pore is intrinsically most stable when closed and that the voltage sensor works to open the pore during depolarization (11, 12). Results from an alanine/valine scan of residues across the entire Shaker pore, by single point mutation, showed that most mutations made the channel easier to open and steepened the response of the channel to changes in voltage. It was argued that, because most mutations likely destabilize protein packing, the closed conformation must be the stable state; this is consistent with the observed crystal structures of Shaker-related channels KcsA and MthK, in the closed and open states, respectively, wherein more optimally and tightly packed helices were seen in the closed conformation (13–15).Because of presumed shared architecture of the gate between HCN and Shaker channels, HCN channels might also be most stable when closed, and thus the voltage sensor would work to open the pore upon hyperpolarization. To test this hypothesis, we performed an alanine/valine scan of the C-terminal 22 amino acids of the S6 segment in HCN2, used as a prototype, and examined pore energetics as described previously in Shaker (11). Choice of this region for mutation was based on: 1) in Shaker, the corresponding region harbors one of two clusters of gating-sensitive residues and 2) it contains the voltage-controlled gate. Surprisingly, the effects of the mutations on channel opening and on the steepness of the channel''s response to voltage are mixed and smaller than those in Shaker. These findings imply that, in HCN2, the stabilities of the open and closed pore are similar, the interactions between the pore and voltage sensor, both structural and functional, are weaker than in Shaker, and that the voltage sensor must apply force to the pore to close it. Thus, Shaker is closed and HCN2 is open in the absence of input from the voltage sensor. Moreover, cAMP binding to the HCN2 channel heightens the effects of the mutations, indicating stronger interactions between the pore and voltage sensor, and tips the energetic balance toward a more stable open state.     

External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K⁺ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K⁺ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd²⁺ and protons on Arabidopsis thaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd²⁺ at 300 µM depolarizes the V₅₀ of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V₅₀ by 49 mV. Regulation of KAT1 by Cd²⁺ is state dependent and consistent with Cd²⁺ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd²⁺ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd²⁺ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K⁺ channels.     

Probing α-310 Transitions in a Voltage-Sensing S4 Helix

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 3₁₀ conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 3₁₀ conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 3₁₀ conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-3₁₀ helical conversions of the S4 in Ci-VSP associated with voltage activation.     

Amino acid pair- and triplet-wise groupings in the interior of α-helical segments in proteins

A statistical approach has been applied to analyse primary structure patterns at inner positions of α-helices in proteins. A systematic survey was carried out in a recent sample of non-redundant proteins selected from the Protein Data Bank, which were used to analyse α-helix structures for amino acid pairing patterns. Only residues more than three positions apart from both termini of the α-helix were considered as inner. Amino acid pairings i, i+k (k=1, 2, 3, 4, 5), were analysed and the corresponding 20×20 matrices of relative global propensities were constructed. An analysis of (i, i+4, i+8) and (i, i+3, i+4) triplet patterns was also performed. These analysis yielded information on a series of amino acid patterns (pairings and triplets) showing either high or low preference for α-helical motifs and suggested a novel approach to protein alphabet reduction. In addition, it has been shown that the individual amino acid propensities are not enough to define the statistical distribution of these patterns. Global pair propensities also depend on the type of pattern, its composition and orientation in the protein sequence. The data presented should prove useful to obtain and refine useful predictive rules which can further the development and fine-tuning of protein structure prediction algorithms and tools.     

High-resolution structural profile of hylaseptin-4: Aggregation,membrane topology and pH dependence of overall membrane binding process

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH₂) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d₃₈ micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.     

Modification of hERG1 channel gating by Cd2+

Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd²⁺ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd²⁺ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd²⁺ shifted the half-point (V_1/2) for the voltage dependence of the OFF gating charge–voltage (Q_OFF-V) relationship with an EC₅₀ of 171 µM; at 0.3 mM, V_1/2 was shifted by +50 mV. Cd²⁺ also induced an as of yet unrecognized small outward current (I_Cd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd²⁺ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd²⁺ in an outward direction to produce I_Cd-out. Co²⁺, Zn²⁺, and La³⁺ at concentrations that caused ∼+35-mV shifts in the Q_OFF-V relationship did not induce a current similar to I_Cd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd²⁺. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd²⁺.     

Accommodation of a D-Phe residue into a right-handed 310-helix: Structure of Boc-D-Phe-(Aib)4-Gly-L-Leu-(Aib)2-Ome,an analogue of the amino terminal segment of antiamoebins and emerimicins

The crystal structure of the nonapeptide Boc-D -Phe-Aib-Aib-Aib-Aib-Gly-Leu-Aib-AibOMe (I), which is an analogue of the N-terminal sequence of antiamoebins and emerimicins, establishes a completely 3₁₀-helical conformation with seven successive intramolecular 4 → 1 hydrogen bonds. The average, ?, ψ values for residues 1–8 are ?59° and ?32°, respectively. Crystal parameters are C₄₇H₇₇N₉O₁₂, space group P1, a = 10.636(4) Å, b = 11.239(4) Å, c = 12.227(6) Å, α = 101.17(4)°, β = 97.22(4)°, γ = 89.80(3)°, Z = 1, R = 5.95% for 3018 data with |F₀| > 3α(F), resolution 0.93 Å. The use of the torsion angle κ = C(i ? 1)N(i)C^α(i)C^β(i), where κ = 68° for D -Phe and κ = 164° for L -Leu, confirms the opposite configurations of these residues. The ?, ψ values of ?62° and ?32° at D -Phe are unusual, since this region is characteristic of residues with L configurations. Peptide I possesses only two chiral residues of opposing configuration. The observed right-handed 3₁₀-helical structure suggests that helix sense has probably been determined by the stereo-chemical preferences of the Leu residue. © 1993 John Wiley & Sons, Inc.