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PubMed2000

A new look at the microtubule binding patterns of dimeric kinesins.

The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.

Adenosine Triphosphate↗

PubMed2001

Crystal structures of neuronal squid Sec1 implicate inter-domain hinge movement in the release of t-SNAREs.

Sec1 molecules associate with t-SNAREs from the syntaxin family in a heterodimeric complex that plays an essential role in vesicle transport and membrane fusion. Neuronal rat n-Sec1 has an arch-shaped three-domain structure, which binds syntaxin 1a through contacts in domains 1 and 3. In both rat nSec1 and homologous squid s-Sec1, a potential effector-molecule binding-pocket is shaped by residues from domains 1 and 2 and is localized on the opposite side of the syntaxin 1a interaction site. Comparison of several crystal forms of unliganded neuronal squid Sec1 indicates a hinge region between domains 1 and 2 which allows domain 1 to rotate along a central axis. This movement could release syntaxin 1a upon interaction with a yet unspecified Sec1 effector molecule(s). The binding of an effector protein may also directly affect the conformation of the helical hairpin of domain 3, which contributes the other significant syntaxin 1a binding sites in the rat nSec1/syntaxin 1a complex structure but adopts multiple conformations in the unliganded s-Sec1 structures reported here.

Amino Acid Sequence↗

PubMed2001

Dynamics and cooperativity of microtubule decoration by the motor protein kinesin.

We describe a theoretical and experimental analysis of the interaction between microtubules and dimeric motor proteins (kinesin, NCD), with special emphasis on the stoichiometry of the interaction, cooperative effects, and their consequences for the interpretation of biochemical and image reconstruction results. Monomeric motors can bind equivalently to microtubules without interference, at a stoichiometry of one motor head per tubulin subunit (alphabeta-heterodimer). By contrast, dimeric motors can interact with stoichiometries ranging between one and two heads per tubulin subunit, depending on binding constants of the first head and the subsequent binding of the second head, and the concentration of dimers in solution. Further, we show that an attractive interaction between the bound motor molecules can explain the higher periodicities observed in decorated microtubules (e.g. 16 nm periodicity), and the non-uniform decoration of a population of microtubules and give an estimate of the strength of this interaction.

Allosteric Site↗

PubMed2001

Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane.

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.

Animals↗

PubMedDate not supplied

Projection structure of an invertebrate rhodopsin.

Rhodopsin is the G-protein-coupled membrane receptor that initiates the visual transduction cascade in retinal photoreceptors. In the present study rhodopsin from the dark-adapted retinas of squid (Loligo forbesi) was detergent-extracted, purified, and reconstituted into native squid photoreceptor lipids following proteolytic cleavage of its prolinerich C-terminus. Two-dimensional crystals of C-terminally truncated rhodopsin reconstituted from octyl glucoside solution formed in a p222(1) lattice (a = 44 A, b = 131 A). Electron micrographs of frozen-hydrated crystals were processed and a projection structure to 8 A resolution was calculated. The projection map obtained is very similar to maps previously determined for bovine and frog rhodopsins although the crystal packing of the molecules is quite different. Comparison of the maps shows that the arrangement of alpha-helices in the proteins is very similar despite their great phylogenetic distance; this structure is likely to be present in the whole superfamily of G-protein-coupled receptors. Invertebrate rhodopsins have a large insertion in the helix 5-helix 6 loop. Assignment of an additional density in the squid rhodopsin map to this region supports a previously proposed helix assignment and identifies the end-to-end contacts as helices 1 and 5.

Animals↗

PubMed1999

Structural comparison of cephalopod hemocyanins: phylogenetic significance.

Hemocyanins, the respiratory molecules of cephalopod mollusks, are hollow cylinders with five internal arches. Three hemocyanins representative of three orders of cephalopods (Benthoctopus species, Octopoda; Vampyroteuthis infernalis, Vampyromorpha; Sepia officinalis, Sepioidea) were subjected to cryoelectron microscopy and three-dimensional (3D) reconstruction. The structure of Benthoctopus hemocyanin, solved at 26.4-A resolution, possesses arches comprising two identical functional units. The similarity between these functional units and the structure recently observed in X-ray crystallography for Octopus by Cuff et al. (J. Mol. Biol., 1998, 232, 522-529) allows the identification of their N- and C-terminal domains in the 3D reconstruction volume. Conversely, arches present in the 3D reconstruction volume of Sepia hemocyanin (21.8 A resolution) contain four functional units that are disposed differently. The strong resemblance between the reconstruction volumes of Vampyroteuthis (21.4-A resolution) and Benthoctopus hemocyanins suggests that Sepioidea diverged from a group containing Octopoda and Vampyromorpha.

Animals↗

PubMed1995

A simple model of K+ channel activation in nerve membrane.

A model is proposed for activation of potassium ion channel current, IK, in squid giant axons, which consists of two closed states and one open state. The rate parameter in the forward direction between the two closed states depends upon previous history. That is, it relaxes exponentially to its steady-state value appropriate to the membrane potential of a voltage clamp step rather than change instantaneously as in traditional models of channel gating. The model successfully describes both the enhancement of the delay in activation of IK with relatively negative prepulse potentials, i.e. the Cole-Moore effect, and the time-dependent rising phase of "on" gating current, which has been reported recently for several types of potassium channels.

Animals↗

PubMed1999

On the role of subthreshold dynamics in neuronal signaling.

The role of subthreshold dynamics in neuronal signaling is examined using periodic pulse train stimulation of the Fitzhugh-Nagumo (FN) model of nerve membrane excitability and results from the squid giant axon as an experimental data base. For a broad range of stimulus conditions the first pulse in a pulse train elicited an action potential, whereas all subsequent pulses elicited subthreshold responses, both in the axon and in the FN model. These results are not well described by the Hodgkin and Huxley 1952 model. Various different patterns of subthreshold responses, including chaotic dynamics, can be observed in both systems-the FN model and the axon-depending upon stimulus conditions. For some conditions action potentials are occasionally interspersed among the subthreshold events with randomly occurring interspike intervals. The randomness is directly attributable to the underlying subthreshold chaos-deterministic chaos-rather than to a stochastic noise source. We conclude that this mechanism may contribute to multimodal interspike interval histograms which have been observed from individual neurons throughout the nervous system.

Action Potentials↗

PubMed2000

A finite-element model for the mechanical analysis of skeletal muscles.

In the present paper, a finite-element model for simulating muscle mechanics is described. Based on nonlinear continuum mechanics an algorithm is proposed that includes the contractile active and passive properties of skeletal muscle. Stress in the muscle is assumed to result from the superposition of a passive and an active part. The passive properties are described by a hyperelastic constitutive material law whereas the active part depends on the fibre length, shortening velocity and an activation function. The constraint of approximate incompressibility of the muscle element is satisfied as a property of the constitutive equations. Because of the nonlinear behaviour of the material and the highly dynamical performance an incremental procedure including iterative methods is used. The advantage of the model over previous formulations is the possibility to integrate the element into an engineering standard finite-element programme ANSYS using advanced numerical tools. The model allows simulations of muscle recruitment, calculations of stress and strain distributions and predictions of muscle shape. Other possible applications are studies of the muscle architecture, the effect of inertia and impacts. First, simple examples are presented.

Animals↗

PubMed2000

Phylogeny and historical biogeography of the loliginid squids (Mollusca: cephalopoda) based on mitochondrial DNA sequence data.

The cephalopod taxon Loliginidae (Cephalopoda: Myopsida) is a species-rich group of tropical and temperate shallow-water squids, many of which are commercial fisheries objects and neurophysiological research organisms. The worldwide distribution of these squids could make Loliginidae a useful case study in shallow-water marine biogeography, but the phylogeny of the group is unknown. To clarify loliginid phylogeny, regions of two mitochondrial genes (the 16S rRNA and the cytochrome c oxidase subunit I genes) were sequenced for members of 19 loliginid species and several outgroups. Maximum-parsimony and maximum-likelihood analyses were performed on a combined data set, as well as on each data set individually. Analyses of the combined data support loliginid monophyly and reveal four clades-one consisting primarily of species in American waters from two genera, one composed of 3 east Atlantic species, one consisting of the bioluminescent loliginids (Uroteuthis sensu Vecchione et al., 1998) plus Loliolus japonica, and one represented by a Loligo (Alloteuthis) subulata-Lolliguncula mercatoris pair. The likelihood of the unconstrained maximum-likelihood tree is not significantly better than the likelihoods of the best trees constrained to Sepioteuthis monophyly or Uroteuthis monophyly, but there is significant support for Lolliguncula polyphyly. Tests of alternative hypotheses of loliginid cladogenesis suggest that cladogenesis within Loliginidae is correlated with the widening of the Atlantic and the closure of the Tethys Sea, although dispersal from the Indo-West Pacific is a reasonable explanation for the origin of the clade of American loliginines.

Animals↗

PubMed1997

Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata).

Adenosine Monophosphate↗

PubMed2001

High-yield expression, purification, and characterization of the recombinant diisopropylfluorophosphatase from Loligo vulgaris.

Organophosphate degrading enzymes are of great interest in light of their ability to detoxify chemical warfare agents. The diisopropylfluorophosphatase (DFPase) from Loligo vulgaris is characterized by its high stability and broad substrate specifity. Here we report the production of large amounts of active, recombinant DFPase using an Escherichia coli expression system. The enzyme was purified to homogeneity using a combination of immobilized metal affinity and ion exchange chromatography. CD-spectroscopy indicates a well folded protein with a high amount of beta-sheet structure. Limited proteolysis was used to gain a deeper insight into the structural organization of the protein. DFPase possesses an internal protease-sensitive region located between amino acids 146 and 149. The two proteolytic fragments remain as a tight complex retaining a DFPase activity comparable to the intact enzyme. Overexpression clones for each fragment were constructed with the expression resulting in the formation of inclusion bodies. Upon isolation and refolding active protein is only formed when both fragments are present. Thus, the two proteolytic fragments are probably part of a stable single-domain protein with multiple contacts between them and only one protease accessible surface loop.

Animals↗

PubMed2006