[Show abstract][Hide abstract] ABSTRACT: We describe the identification in aphids of a unique heterodimeric voltage-gated sodium channel which has an atypical ion selectivity filter and, unusually for insect channels, is highly insensitive to tetrodotoxin. We demonstrate that this channel has most likely arisen by adaptation (gene fission or duplication) of an invertebrate ancestral mono(hetero)meric channel. This is the only identifiable voltage-gated sodium channel homologue in the aphid genome(s), and the channel’s novel selectivity filter motif (DENS instead of the usual DEKA found in other eukaryotes) may result in a loss of sodium selectivity, as indicated experimentally in mutagenised Drosophila channels.
[Show abstract][Hide abstract] ABSTRACT: We describe a scalable artificial bilayer lipid membrane platform for rapid electrophysiological screening of ion channels and transporters. A passive pumping method is used to flow microliter volumes of ligand solution across a suspended bilayer within a microfluidic chip. Bilayers are stable at flow rates up to ∼0.5 μl/min. Phospholipid bilayers are formed across a photolithographically defined aperture made in a dry film resist within the microfluidic chip. Bilayers are stable for many days and the low shunt capacitance of the thin film support gives low-noise high-quality single ion channel recording. Dose-dependent transient blocking of α-hemolysin with β-cyclodextrin (β-CD) and polyethylene glycol is demonstrated and dose-dependent blocking studies of the KcsA potassium channel with tetraethylammonium show the potential for determining IC50 values. The assays are fast (30 min for a complete IC50 curve) and simple and require very small amounts of compounds (100 μg in 15 μl). The technology can be scaled so that multiple bilayers can be addressed, providing a screening platform for ion channels, transporters, and nanopores.
[Show abstract][Hide abstract] ABSTRACT: The favourable transfer free energy for a transmembrane (TM) α-helix between the aqueous phase and lipid bilayer underlies the stability of membrane proteins. However, the connection between the energetics and process of membrane protein assembly by the Sec61/SecY translocon complex in vivo is not clear. Here, we directly determine the partitioning free energies of a family of designed peptides using three independent approaches: an experimental microsomal Sec61 translocon assay, a biophysical (spectroscopic) characterization of peptide insertion into hydrated planar lipid bilayer arrays, and an unbiased atomic-detail equilibrium folding-partitioning molecular dynamics simulation. Remarkably, the measured free energies of insertion are quantitatively similar for all three approaches. The molecular dynamics simulations show that TM helix insertion involves equilibrium with the membrane interface, suggesting that the interface may play a role in translocon-guided insertion.
[Show abstract][Hide abstract] ABSTRACT: The cationic amphipathic designer peptide LAH4 exhibits potent antimicrobial, nucleic acid transfection and cell penetration activities. Closely related derivatives have been developed to enhance viral transduction for gene therapeutic assays. LAH4 contains four histidines and, consequently, its overall charge and membrane topology in lipid bilayers are strongly pH dependent. In order to better understand the differential interactions of this amphipathic peptide with negatively-charged membranes its interactions, topologies, and penetration depth were investigated in the presence of lipid bilayers as a function of pH, buffer, phospholipid head group, and fatty acyl chain composition using a combination of oriented synchrotron radiation circular dichroism spectroscopy as well as oriented and non-oriented solid-state NMR spectroscopy. This combination of methods indicates that in the presence of lipids with phosphatidylglycerol head groups, the topological equilibria of LAH4 is shifted towards more in-plane configurations even at neutral pH. In contrast, a transmembrane alignment is promoted when LAH4 interacts with membranes made of dimyristoyl phospholipids rather than palmitoyl-oleoyl-phospholipids. Finally, the addition of citrate buffer favours LAH4 transmembrane alignments, even at low pH, probably by complex formation with the cationic charges of the peptide. In summary, this study has revealed that the membrane topology of this peptide is readily modulated by the environmental conditions.
European Biophysics Journal 09/2014; 43(10-11). · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circular dichroism (CD) spectroscopy is a valuable method for defining canonical secondary structure contents of proteins based on empirically-defined spectroscopic signatures derived from proteins with known three-dimensional structures. Many proteins identified as being “Intrinsically Disordered Proteins” have a significant amount of their structure that is neither sheet, helix, nor turn; this type of structure is often classified by CD as “other”, “random coil”, “unordered” or “disordered”. However the “other” category can also include polyprolineII (PPII)-type structures, whose spectral properties have not been well-distinguished from those of unordered structures.In this study, synchrotron radiation circular dichroism spectroscopy was used to investigate the spectral properties of collagen and polyproline, which both contain PPII-type structures. Their native spectra were compared as representatives of PPII structures. In addition, their spectra before and after treatment with various conditions to produce unfolded or denatured structures were also compared, with the aim of defining the differences between CD spectra of PPII and disordered structures. We conclude that the spectral features of collagen are more appropriate than those of polyproline for use as the representative spectrum for PPII structures present in typical amino acid-containing proteins, and that the single most characteristic spectroscopic feature distinguishing a PPII structure from a disordered structure is the presence of a positive peak around 220 nm in the former but not in the latter. These spectra are now available for inclusion in new reference data sets used for CD analyses of the secondary structures of soluble proteins.
[Show abstract][Hide abstract] ABSTRACT: Diacylglycerol acyltransferase 1 (DGAT1) is a microsomal membrane enzyme responsible for the final step in the synthesis of triacylglycerides. Although DGATs from a wide range of organisms have nearly identical sequences, there is little structural information available for these enzymes. The substrate binding sites of DGAT1 are predicted to be in its large luminal extramembranous loop and to include common motifs with acyl-CoA cholesterol acyltransferase enzymes and the diacylglycerol binding domain found in protein kinases. In this study, synthetic peptides corresponding to the predicted binding sites of DGAT1 enzyme were examined using synchrotron radiation circular dichroism spectroscopy, fluorescence emission and adsorption onto lipid monolayers to determine their interactions with substrates associated with triacylglyceride synthesis (oleoyl-CoA and dioleoylglycerol). One of the peptides, Sit1, which includes the FYxDWWN motif common to both DGAT1 and acyl-CoA cholesterol acyltransferase, changes its conformation in the presence of both substrates, suggesting its capability to bind their acyl chains. The other peptide (Sit2), which includes the putative diacylglycerol binding domain HKWCIRHFYKP found in protein kinase C and diacylglycerol kinases, appears to interact with the charged headgroup region of the substrates. Moreover, in an extended-peptide which contains Sit1 and Sit2 sequences separated by a flexible linker, larger conformational changes were induced by both substrates, suggesting the two binding sites may bring the substrates into close proximity within the membrane, thus catalysing the formation of the triacylglyceride product.
[Show abstract][Hide abstract] ABSTRACT: Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in dif-ferent human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the pro-karyotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Na v 1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs. crystal structure | pharmacology N ine highly homologous human voltage-gated sodium chan-nel isoforms have been identified (1). They are composed of single polypeptide chains containing four pseudorepeated domains (designated DI to DIV), each of which is composed of six transmembrane helical segments (S1 to S6); the pore region is formed from S5 to S6, including the intervening loop and se-lectivity filter (SF), from all four domains. Prokaryotic sodium channels, in contrast, are homotetramers of four identical poly-peptide chains, each of which is equivalent to, and homologous with, one of the eukaryotic domains. Although there are as yet no crystal structures of eukaryotic sodium channels, crystal structures of several prokaryotic sodium channels in different conformational states have been determined, including ones with closed (2), partially (3) and fully (4) open pores, and two po-tentially inactivated forms (5, 6). Mutations in human sodium channels (hNa v s) have been linked to channelopathies such as epilepsy, cardiac arrhythmia, and chronic pain syndromes; con-sequently sodium channel blockers have been developed as anti-convulsant, antiarrhythmic, and local anesthetic drugs (7–10). Several eukaryotic calcium channel blocker drugs have pre-viously been found to bind and block prokaryotic sodium chan-nels (11–13). Results
Proceedings of the National Academy of Sciences 06/2014; 111(23). · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.
[Show abstract][Hide abstract] ABSTRACT: S100A12 (Calgranulin C) is a small acidic calcium-binding peripheral membrane protein with two EF-hand structural motifs. It is expressed in macrophages and lymphocytes and highly up-regulated in several human inflammatory diseases. In pigs, S100A12 is abundant in the cytosol of granulocytes, where it is believed to be involved in signal modulation of inflammatory process. In this study, we investigated the interaction of the porcine S100A12 with phospholipid bilayers and the effect that ions (Ca(2+), Zn(2+) or both together) have in modifying protein-lipid interactions. More specifically, we intended to address issues such as: (1) is the protein-membrane interaction modulated by the presence of ions? (2) is the protein overall structure affected by the presence of the ions and membrane models simultaneously? (3) what are the specific conformational changes taking place when ions and membranes are both present? (4) does the protein have any kind of molecular preferences for a specific lipid component? To provide insight into membrane interactions and answer those questions, synchrotron radiation circular dichroism spectroscopy, fluorescence spectroscopy, and surface plasmon resonance were used. The use of these combined techniques demonstrated that this protein was capable of interacting both with lipids and with ions in solution, and enabled examination of changes that occur at different levels of structure organization. The presence of both Ca(2+) and Zn(2+) ions modify the binding, conformation and thermal stability of the protein in the presence of lipids. Hence, these studies examining molecular interactions of porcine S100A12 in solution complement the previously determined crystal structure information on this family of proteins, enhancing our understanding of its dynamics of interaction with membranes.
PLoS ONE 12/2013; 8(12):e82555. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circular dichroism (CD) spectroscopy is a widely used technique for the charac-terisation of proteins. A number of CD instruments are currently on the market, and there are more than a dozen synchrotron radiation circular dichroism (SRCD) beamlines in operation worldwide. All produce different output formats and contents. In order for users of CD and SRCD data to be able simply to compare and contrast data and the associated recorded or unrecorded metadata, it is essential to have a common data format. For this reason, the JCAMP-DX-CD format for CD spectroscopy has been developed, based on extensive con-sultations with users and senior representatives of all the instrument manufacturers and beamlines, and under the auspices of IUPAC, based on the Joint Committee on Atomic and Physical Data Exchange protocols. The availability of a common format is also important for deposition to, and access from, the Protein Circular Dichroism Data Bank, the public repos-itory for CD and SRCD data and metadata. The JCAMP-DX-CD format can be read by stan-dard JCAMP programs such as JSpecView. We have also created a series of parsers, avail-able at the DichroJCAMP web site (http://valispec.cryst.bbk.ac.uk/formatConverter/ dichroJCAMPDX-CD.html), which will enable the conversion between instrument and beamline formats and the JCAMP-DX-CD format.
[Show abstract][Hide abstract] ABSTRACT: Voltage-gated sodium channels have essential roles in electrical signalling. Prokaryotic sodium channels are tetramers consisting of transmembrane (TM) voltage-sensing and pore domains, and a cytoplasmic carboxy-terminal domain. Previous crystal structures of bacterial sodium channels revealed the nature of their TM domains but not their C-terminal domains (CTDs). Here, using electron paramagnetic resonance (EPR) spectroscopy combined with molecular dynamics, we show that the CTD of the NavMs channel from Magnetococcus marinus includes a flexible region linking the TM domains to a four-helix coiled-coil bundle. A 2.9 Å resolution crystal structure of the NavMs pore indicates the position of the CTD, which is consistent with the EPR-derived structure. Functional analyses demonstrate that the coiled-coil domain couples inactivation with channel opening, and is enabled by negatively charged residues in the linker region. A mechanism for gating is proposed based on the structure, whereby splaying of the bottom of the pore is possible without requiring unravelling of the coiled-coil.
[Show abstract][Hide abstract] ABSTRACT: Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria.
The ß-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the E.coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined.
Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold.
The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method. General Significance We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.
Biochimica et Biophysica Acta 08/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The micro-exon genes (MEG) of Schistosoma mansoni, a parasite responsible for the second most widely spread tropical disease, code for small secreted proteins with sequences unique to the Schistosoma genera. Bioinformatics analyses suggest the soluble domain of the MEG-14 protein will be largely disordered, and using synchrotron radiation circular dichroism spectroscopy, its secondary structure was shown to be essentially completely unfolded in aqueous solution. It does, however, show a strong propensity to fold into more ordered structures under a wide range of conditions. Partial folding was produced by increasing temperature (in a reversible process), contrary to the behavior of most soluble proteins. Furthermore, significant folding was observed in the presence of negatively charged lipids and detergents, but not in zwitterionic or neutral lipids or detergents. Absorption onto a surface followed by dehydration stimulated it to fold into a helical structure, as it did when the aqueous solution was replaced by nonaqueous solvents. Hydration of the dehydrated folded protein was accompanied by complete unfolding. These results support the identification of MEG-14 as a classic intrinsically disordered protein, and open the possibility of its interaction/folding with different partners and factors being related to multifunctional roles and states within the host.
[Show abstract][Hide abstract] ABSTRACT: Circular dichroism (CD) spectroscopy is widely used in structural biology as a technique for examining the structure, folding and conformational changes of proteins. A new server, ValiDichro, has been developed for checking the quality and validity of CD spectral data and metadata, both as an aid to data collection and processing and as a validation procedure for spectra to be included in publications. ValiDichro currently includes 25 tests for data completeness, consistency and quality. For each test that is done, not only is a validation report produced, but the user is also provided with suggestions for correcting or improving the data. The ValiDichro server is freely available at http://valispec.cryst.bbk.ac.uk/circularDichroism/ValiDichro/upload.html.
Nucleic Acids Research 04/2013; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.
Biochemical and Biophysical Research Communications 04/2013; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The pyrethroid insecticides are a very successful group of compounds that target invertebrate voltage-gated sodium channels and are widely used in the control of insects, ticks and mites. It is well established that some pyrethroids are good insecticides whereas others are more effective as acaricides. This species specificity is advantageous for controlling particular pest(s) in the presence of another non-target invertebrate, for example controlling the Varroa mite in honey bee colonies. RESULTS: We applied in-silico techniques to compare the voltage-gated sodium channels of insects versus ticks and mites and their interactions with a range of pyrethroids and DDT analogues. We identified a single amino acid difference within the pyrethroid binding pocket of ticks/mites that may have significant impact on the effectiveness of pyrethroids as acaricides. Other individual amino acid differences within the binding pocket in distinct tick and mite species may provide a basis for future acaricidal selectivity. CONCLUSIONS: 3D modelling of the pyrethroid/DDT receptor site has led to a new hypothesis to explain the preferential binding of acaricidal pyrethroids to the sodium channels of ticks/mites. This is important for understanding pyrethroid selectivity and the potential effects of mutations that can give rise to resistance to pyrethroids in commercially-important pest species.
Pest Management Science 04/2013; 70(3). · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative (22)Na(+) uptake into proteoliposomes containing a 3∶1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different "guest" glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains.
PLoS ONE 04/2013; 8(4):e61216. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channel's full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate.
Proceedings of the National Academy of Sciences 03/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The membrane interactions of the antimicrobial peptides aurein 1.2 and caerin 1.1 were observed by (31)P and (2)H solid-state NMR and circular dichroism spectroscopy. Both peptides were relatively unstructured in water. In the presence of dimyristoylphosphatidylcholine (DMPC) and mixed DMPC and dimyristoylphosphatidylglycerol (DMPG) vesicles, both peptides displayed a considerable increase in helical content with the shorter aurein peptide having a higher α-helix content in both lipid systems. In fluid phase DMPC vesicles, the peptides displayed differential interactions: aurein 1.2 interacted primarily with the bilayer surface, while the longer caerin 1.1 was able to penetrate into the bilayer interior. Both peptides displayed a preferential interaction with the DMPG component in DMPC/DMPG bilayers, with aurein 1.2 limited to interaction with the surface and caerin 1.1 able to penetrate into the bilayer and promote formation of a mixture of lipid phases or domains. In gel phase DMPC vesicles, aurein 1.2 disrupted the bilayer apparently through a carpet mechanism, while no additional interaction was seen with caerin 1.1. Although a lamellar bilayer was retained with the mixed DMPC/DMPG vesicles below the phase transition, both caerin 1.1 and aurein 1.2 promoted disruption of the bilayer and formation of an isotropic phase. The peptide interaction was enhanced relative to the fluid phase and was likely driven by co-existence of membrane defects. This study thus demonstrates that the effects of the lipid phase and domains need to be considered when studying membrane interactions of antimicrobial peptides.
Biochimica et Biophysica Acta 03/2013; 1828:1868-72. · 4.66 Impact Factor