[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; · 8.28 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; · 2.41 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; · 2.59 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; · 9.74 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
[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 01/2013; 8(4):e61216. · 3.73 Impact Factor
[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 01/2013; 8(12):e82555. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Voltage-gated sodium channels are vital membrane proteins essential for electrical signalling; in humans, they are key targets for the development of pharmaceutical drugs. Here we report the crystal structure of an open-channel conformation of NavMs, the bacterial channel pore from the marine bacterium Magnetococcus sp. (strain MC-1). It differs from the recently published crystal structure of a closed form of a related bacterial sodium channel (NavAb) by having its internal cavity accessible to the cytoplasmic surface as a result of a bend/rotation about a central residue in the carboxy-terminal transmembrane segment. This produces an open activation gate of sufficient diameter to allow hydrated sodium ions to pass through. Comparison of the open and closed structures provides new insight into the features of the functional states present in the activation cycles of sodium channels and the mechanism of channel opening and closing.
[show abstract][hide abstract] ABSTRACT: The Protein Circular Dichroism Data Bank (PCDDB) is a web-based resource containing circular dichroism (CD) and synchrotron radiation circular dichroism spectral and associated metadata located at http://pcddb.cryst.bbk.ac.uk. This resource provides a freely available, user-friendly means of accessing validated CD spectra and their associated experimental details and metadata, thereby enabling broad usage of this material and new developments across the structural biology, chemistry, and bioinformatics communities. The resource also enables researchers utilizing CD as an experimental technique to have a means of storing their data at a secure site from which it is easily retrievable, thereby making their results publicly accessible, a current requirement of many grant-funding agencies world-wide, as well as meeting the data-sharing requirements for journal publications. This tutorial provides extensive information on searching, accessing, and downloading procedures for those who wish to utilize the data available in the data bank, and detailed information on deposition procedures for creating and validating entries, including comprehensive explanations of their contents and formats, for those who wish to include their data in the data bank.
[show abstract][hide abstract] ABSTRACT: Neonicotinoid insecticides target nicotinic acetylcholine receptors (nAChR) in the nervous system of insects but are largely ineffective against ticks. This study aimed to identify the molecular basis for this insensitivity. A homology model of the nAChR binding domain was generated on the basis of the crystal structure of an acetylcholine-binding protein with the insecticide imidacloprid bound. We hypothesized that tick β-subunits would differ at a critical residue (Arg81) in their D loops. To test this, we sequenced nAChR genes from five tick species and found that instead of the conserved arginine found in insects, a glutamine was present in all the tick sequences.
[show abstract][hide abstract] ABSTRACT: Circular dichroism (CD) spectroscopy is a widely used method for examining the structure, folding and conformational changes of proteins. A new online CD analysis server (DichroMatch) has been developed for identifying proteins with similar spectral characteristics by detecting possible structurally and functionally related proteins and homologues. DichroMatch includes six different methods for determining the spectral nearest neighbours to a query protein spectrum and provides metrics of how similar these spectra are and, if corresponding crystal structures are available for the closest matched proteins, information on their secondary structures and fold classifications. By default, DichroMatch uses all the entries in the Protein Circular Dichroism Data Bank (PCDDB) for its comparison set, providing the broadest range of publicly available protein spectra to match with the unknown protein. Alternatively, users can download or create their own specialized data sets, thereby enabling comparisons between the structures of related proteins such as wild-type versus mutants or homologues or a series of spectra of the same protein under different conditions. The DichroMatch server is freely available at http://dichromatch.cryst.bbk.ac.uk.
Nucleic Acids Research 05/2012; 40(Web Server issue):W547-52. · 8.28 Impact Factor