Electron and X-ray microscopic analyses of reaggregated materials obtained after fractionation of dissolved sporopollenin

Summary Exines fromTypha angustifolia L. pollen were dissolved in hot 2-aminoethanol. The solubilisate was successively fractionated and reaggregated via a dialysis cascade with dialysis tubings of different exclusion volumina. Four fractions of reaggregated material with different molecular mass were obtained. Fraction 1 with a molecular mass above 25,000 Da, fraction 2 with a molecular mass between 10,000–25,000 Da, fraction 3 with a molecular mass between 5,000–10,000 Da, and fraction 4 of a molecular mass lower than 5,000 Da. The fractions were comparatively analysed by scanning and transmission electron microscopy and X-ray microscopy. The material of the fractions with a molecular mass above 10,000 Da exhibit high congruence to the initial material. Analysis of the reaggregated material with the lowest molecular mass revealed special distinct substructures which in form and size showed high similarities to substructures of exines described in literature. In detail, spherical substructures consisting of an electron-dense core surrounded by an electron-transparent corona and in addition elongated substructures with a distinctive surface sculpture were detected.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy     

Blue copper proteins: a comparative analysis of their molecular interaction properties

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.     

Using known substructures in protein model building and crystallography.

Retinol binding protein can be constructed from a small number of large substructures taken from three unrelated proteins. The known structures are treated as a knowledge base from which one extracts information to be used in molecular modelling when lacking true atomic resolution. This includes the interpretation of electron density maps and modelling homologous proteins. Models can be built into maps more accurately and more quickly. This requires the use of a skeleton representation for the electron density which improves the determination of the initial chain tracing. Fragment-matching can be used to bridge gaps for inserted residues when modelling homologous proteins.     

Toward the understanding of MNEI sweetness from hydration map surfaces

The binding mechanism of sweet proteins to their receptor, a G-protein-coupled receptor, is not supported by direct structural information. In principle, the key groups responsible for biological activity (glucophores) can be localized on a small structural unit (sweet finger) or spread on a larger surface area. A recently proposed model, called "wedge model", implies a large surface of interaction with the receptor. To explore this model in greater detail, it is necessary to examine the physicochemical features of the surfaces of sweet proteins, since their interaction with the receptor, with respect to that of small sweeteners, is more dependent on general physicochemical properties of the interface, such as electrostatic potential and hydration. In this study, we performed exhaustive molecular dynamics simulations in explicit water of the sweet protein MNEI and of its structural mutant G-16A, whose sweetness is one order of magnitude lower than that of MNEI. Solvent density and self-diffusion calculated from molecular dynamics simulations suggest a likely area of interaction delimited by four stretches arranged as a tetrahedron whose shape is complementary to that of a cavity on the surface of the receptor, in agreement with the wedge model. The suggested area of interaction is amazingly consistent with known mutagenesis data. In addition, the asymmetric hydration of the only helix in both proteins hints at a specific role for this secondary structure element in orienting the protein during the binding process.     

How Far Does a Receptor Influence Vibrational Properties of an Odorant?

The biophysical mechanism of the sense of smell, or olfaction, is still highly debated. The mainstream explanation argues for a shape-based recognition of odorant molecules by olfactory receptors, while recent investigations suggest the primary olfactory event to be triggered by a vibrationally-assisted electron transfer reaction. We consider this controversy by studying the influence of a receptor on the vibrational properties of an odorant in atomistic details as the coupling between electronic degrees of freedom of the receptor and the vibrations of the odorant is the key parameter of the vibrationally-assisted electron transfer. Through molecular dynamics simulations we elucidate the binding specificity of a receptor towards acetophenone odorant. The vibrational properties of acetophenone inside the receptor are then studied by the polarizable embedding density functional theory approach, allowing to quantify protein-odorant interactions. Finally, we judge whether the effects of the protein provide any indications towards the existing theories of olfaction.     

A note on the cross-reactivity of antibodies.

The energies associated with the interaction and conformational change of model compounds simulating drug and receptor features have been predicted using CNDO/2 calculations on a united molecule. In this second study of the hypothesis of remote recognition of preferred conformation we have examined systems involved in hydrogen bonding. We have found that a system containing interacting electron lone pairs behaves at variance with our hypothesis. Other systems in this study support the hypothesis.     

Structural and functional insights into the interaction of echoviruses and decay-accelerating factor

Many enteroviruses bind to the complement control protein decay-accelerating factor (DAF) to facilitate cell entry. We present here a structure for echovirus (EV) type 12 bound to DAF using cryo-negative stain transmission electron microscopy and three-dimensional image reconstruction to 16-A resolution, which we interpreted using the atomic structures of EV11 and DAF. DAF binds to a hypervariable region of the capsid close to the 2-fold symmetry axes in an interaction that involves mostly the short consensus repeat 3 domain of DAF and the capsid protein VP2. A bulge in the density for the short consensus repeat 3 domain suggests that a loop at residues 174-180 rearranges to prevent steric collision between closely packed molecules at the 2-fold symmetry axes. Detailed analysis of receptor interactions between a variety of echoviruses and DAF using surface plasmon resonance and comparison of this structure (and our previous work; Bhella, D., Goodfellow, I. G., Roversi, P., Pettigrew, D., Chaudhry, Y., Evans, D. J., and Lea, S. M. (2004) J. Biol. Chem. 279, 8325-8332) with reconstructions published for EV7 bound to DAF support major differences in receptor recognition among these viruses. However, comparison of the electron density for the two virus.receptor complexes (rather than comparisons of the pseudo-atomic models derived from fitting the coordinates into these densities) suggests that the dramatic differences in interaction affinities/specificities may arise from relatively subtle structural differences rather than from large-scale repositioning of the receptor with respect to the virus surface.     

Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP

Using biochemical assays, it has been demonstrated that expression of Ebola virus VP40 alone in mammalian cells induced production of particles with a density similar to that of virions. To determine the morphological properties of these particles, cells expressing VP40 and the particles released from the cells were examined by electron microscopy. VP40 induced budding from the plasma membrane of filamentous particles, which differed in length but had uniform diameters of approximately 65 nm. When the Ebola virus glycoprotein (GP) responsible for receptor binding and membrane fusion was expressed in cells, we found pleomorphic particles budding from the plasma membrane. By contrast, when GP was coexpressed with VP40, GP was found on the filamentous particles induced by VP40. These results demonstrated the central role of VP40 in formation of the filamentous structure of Ebola virions and may suggest an interaction between VP40 and GP in morphogenesis.     

Direct measurement of the interaction force between immunostimulatory CpG-DNA and TLR9 fusion protein

The specific interaction between human Toll-like receptor 9 (TLR9)-ectodomain (ECD)-fusion protein and immunostimulatory CpG-DNA was measured using force spectroscopy. Flexible tethers were used between receptors and surface as well as between DNA and atomic force microscope tip to make efficient recognition studies possible. The molecular recognition forces detected are in the range of 50 to 150 ± 20 pN at the used force-loading rates, and the molecular interaction probability was much reduced when the receptors were blocked with free CpG-DNA. A linear increase of the unbinding force with the logarithm of the loading rate was found over the range 0.1 to 30 nN/s. This indicates a single potential barrier characterizing the energy landscape and no intermediate state for the unbinding pathway of CpG-DNA from the TLR9-ECD. Two important kinetic parameters for CpG-DNA interaction with TLR9-ECD were determined from the force-loading rate dependency: an off-rate of k(off) = 0.14 ± 0.10 s(-1) and a binding interaction length of x(β) = 0.30 ± 0.03 nm, which are consistent with literature values. Various models for the molecular interaction of this innate immune receptor binding to CpG-DNA are discussed.     

Lactoferrin specifically inhibits endocytosis of chylomicron remnants but not alpha-macroglobulin.

Our recently found nonlipoprotein inhibitor of chylomicron remnant uptake, lactoferrin, has been investigated in vivo and in vitro. Lipoprotein lipase extracted triglycerides from chylomicrons, doubly labeled with [3H]retinol/[14C]oleate, in the presence of lactoferrin normally. The subsequent uptake of remnants into liver was retarded considerably. In the intact rat, chylomicron remnants (CRs), predominantly labeled in the apoB48 moiety by 125I, were excluded from the hepatic endosomal compartment in the presence of lactoferrin as shown in subcellular fractionation studies of rat livers. In tissue culture, internalization of [125I]chylomicron remnants was inhibited in the presence of 14 pM lactoferrin by 70%. Upon removal of lactoferrin, internalization was rapidly restored. Protease digestion eliminated the inhibitory effect completely. Modification of arginine residues with cyclohexanedione reversibly removed the inhibitory potency of lactoferrin. We located by molecular modeling an alpha-helical segment in lactoferrin on the exposed surface of the molecule containing the sequence Arg-X-X-Arg-Lys-X-Arg, which resembles the receptor recognition structure in apolipoprotein E (apoE). This firmly established ligand correspondence with apoE, the candidate ligand for CR recognition by the receptor. Finally, the postulated second function of low density lipoprotein receptor-related protein, uptake of alpha-2-macroglobulin (alpha 2M) was found to be distinct from lipoprotein binding, since lactoferrin inhibited CR but not alpha 2M internalization. In addition, CR uptake was not affected by alpha 2M. We conclude that if a bifunctional receptor were to operate, its diverse functions were exerted by independently operating substructures. The results of our in vivo and cell culture experiments are, however, entirely compatible with the existence of two receptors as well.     

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: insight into the ganglioside binding mechanism

Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3′-sialyllactose at a resolution of 3.0 Å. In the structure, an electron density derived from the 3′-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.     

Molecular similarity analysis between insect juvenile hormone and N, N-diethyl-m-toluamide (DEET) analogs may aid design of novel insect repellents

Molecular similarity analysis of stereoelectronic properties between natural insect juvenile hormone (JH), -a synthetic insect juvenile hormone mimic (JH-mimic, undecen-2-yl carbamate), and N, N-diethyl-m-toluamide (DEET) and its analogs reveals similarities that may aid the design of more efficacious insect repellents and give a better insight into the mechanism of repellent action. The study involves quantum chemical calculations using the AM1 semi-empirical computational method enabling a conformational search for the lowest and most abundant energy conformers of JH, JH-mimic, and 15 DEET compounds, followed by complete geometry optimization of the conformers. Similarity analyses of stereoelectronic properties such as structural parameters, atomic charges, dipole moments, molecular electrostatic potentials, and highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies were performed on JH, JH-mimic and the DEET compounds. The similarity of stereoelectronic attributes of the amide/ester moiety, the negative electrostatic potential regions beyond the van der Waals surface, and the large distribution of hydrophobic regions in the compounds appear to be the three important factors leading to a similar interaction with the JH receptor. The similarity of electrostatic profiles beyond the van der Waals surface is likely to play a crucial role in molecular recognition interaction with the JH receptor from a distance. This also suggests electrostatic bioisosterism of the amide group of the DEET compounds and JH-mimic and, thus, a model for molecular recognition at the JH receptor. The insect repellent property of the DEET analogs may thus be attributed to a conflict of complementarity for the JH receptor binding sites.     

Lipid-bound structure of an apolipoprotein E-derived peptide

Apolipoprotein (apo) E regulates plasma lipid homeostasis through its ability to interact with the low density lipoprotein (LDL) receptor family. Whereas apoE is not a ligand for receptor binding in buffer alone, interaction with lipid confers receptor recognition properties. To investigate the nature of proposed lipid binding-induced conformational changes in apoE, we employed multidimensional heteronuclear NMR spectroscopy to determine the structure of an LDL receptor-active, 58-residue peptide comprising residues 126-183 of apoE in association with the micelle-forming lipid dodecylphosphocholine (DPC). In the presence of 34 mm DPC the peptide forms a continuous amphipathic helix from Glu131 to Arg178. NMR relaxation studies of DPC-bound apoE-(126-183), in contrast to apoE-(126-183) in the presence of TFE, are consistent with an isotropically tumbling peptide in solution giving a global correlation time of approximately 12.5 ns. These data indicate that the helical peptide is curved and constrained by a lipid micelle consisting of approximately 48 DPC molecules. Although the peptide behaves as if it were tumbling isotropically, spectral density analysis reveals that residues 150-183 have more motional freedom than residues 134-149. These molecular and dynamic features are discussed further to provide insight into the structural basis for the interaction between apoE and the ligand binding repeats of the LDL receptor.     

Question 9: Quantum Self-Assembly and Photoinduced Electron Tunneling in Photosynthetic Systems of Artificial Minimal Living Cells

Natural and artificial living cells and their substructures are self-assembling, due to electron correlation interactions among biological and water molecules, which lead to attractive dispersion forces and hydrogen bonds. Dispersion forces are weak intermolecular forces that arise from the attractive force between quantum multipoles. A hydrogen bond is a special type of quantum attractive interaction that exists between an electronegative atom and a hydrogen atom bonded to another electronegative atom; and this hydrogen atom exist in two quantum states. The best method to simulate these dispersion forces and hydrogen bonds is to perform quantum mechanical non-local density functional potential calculations of artificial minimal living cells consisting of around 1,000 atoms. The cell systems studied are based on peptide nucleic acid and are 3.0–4.2 nm in diameter. The electron tunneling and associated light absorption of the most intense transitions, as calculated by the time dependent density functional theory method, differs from spectroscopic experiments by only 0.2–0.3 nm, which is within the value of experiment errors. This agreement implies that the quantum mechanically self-assembled structures of artificial minimal living cells very closely approximate realistic ones.     

A molecular theory of recognition and activation at a 5-HT receptor based on a quantum chemical approach to structure activity relationships

The study of structure activity relationships (SAR) is based on the delineation of the causal relationships between the properties of molecules and the observed responses evoked by the interaction of these molecules with biological systems. The methods of theoretical and quantum chemistry describe accurately the molecular properties that are determined by molecular structure and provide a rigorous link between structure and activity. We study the molecular events in the pharmacological mechanism of drugs interacting with the receptor of 5-hydroxytryptamine (5-HT, serotonin) by defining the elements of recognition and by analyzing the changes induced in a molecular model for the receptor. These steps define the relationship between the properties of the drugs and their ability to be recognized and cause the activation of the receptors. Consequently, our quantum chemical studies of drug-receptor interactions explain the selectivity of receptors and the molecular determinants for agonism and antagonism on the 5-HT receptor.     

Identification of residues essential for carbohydrate recognition and cation dependence of the 46-kDa mannose 6-phosphate receptor

The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays an essential role in the biogenesis of lysosomes by diverting newly synthesized mannose 6-phosphate (Man-6-P)-containing lysosomal enzymes from the secretory pathway to acidified endosomes. Previous crystallographic studies of the CD-MPR have identified 11 amino acids within its carbohydrate binding pocket. These residues were evaluated quantitatively by assaying the binding affinity of mutant receptors containing a single amino acid substitution toward a lysosomal enzyme. The results show that substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 results in a >800-fold decrease in affinity, demonstrating these four amino acids are essential for carbohydrate recognition by the CD-MPR. Solution binding and surface plasmon resonance analyses demonstrated that the presence of Mn2+ enhanced the affinity of the CD-MPR for a lysosomal enzyme by 2- to 4-fold and increased the stoichiometry of the interaction between a heterogeneous population of a lysosomal enzyme and the receptor by approximately 3-fold. In contrast, substitution of Asp-103 results in a protein that no longer exhibits enhanced binding affinities or altered stoichiometry in the presence of cations, and electron spin resonance demonstrated that the D103S mutant exhibits a 6-fold lower affinity for Mn2+ than the wild-type receptor (Kd = 3.7 6 1.4 mM versus 0.6 6 0.1 mM). Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers. Taken together, these studies provide the molecular basis for high affinity carbohydrate recognition by the CD-MPR. Furthermore, Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD-MPR.     

Antibody recognition imaging by force microscopy.

We have developed a method that combines dynamic force microscopy with the simultaneous molecular recognition of an antigen by an antibody, during imaging. A magnetically oscillated atomic force microscopy tip carrying a tethered antibody was scanned over a surface to which lysozyme was bound. By oscillating the probe at an amplitude of only a few nanometers, the antibody was kept in close proximity to the surface, allowing fast and efficient antigen recognition and gentle interaction between tip and sample. Antigenic sites were evident from reduction of the oscillation amplitude, as a result of antibody-antigen recognition during the lateral scan. Lysozyme molecules bound to the surface were recognized by the antibody on the scanning tip with a few nanometers lateral resolution. In principle, any ligand can be tethered to the tip; thus, this technique could potentially be used for nanometer-scale epitope mapping of biomolecules and localizing receptor sites during biological processes.     

生命机体危险因子信息特征分析

旨在生命机体与危险因子间建立生命物质信息对话平台,以物质化的信息,或信息化的物质的思维进行理解和推论,建立物质承载信息,信息通过物质的形式传递的思维模式,对生命信息中危险因子的属性、分类以及生命机体对危险因子的识别方式进行分析,从以上两个方面不同角度阐述危险因子与生命机体机制信息的相关互动关系。在分析与解读的过程中,主张建立合乎自然生成逻辑的科学概念,而摒弃一些主观是非逻辑所推导建立起来的概念,还原生命物质和生命过程的本来生物学位置及性质;主张一元化的思考方式和整体性理解生命信息安全控制机制的核心部分。分析结果:就生命信息中危险因子的属性和分类进行了分析,指出危险因子具有生命信息属性和生命安全属性两个属性,依据危险因子物质来源的不同,分外源性和内源性两类,绘出危险因子来源分类汇总图;从机体方面对危险因子识别方式不同,分为PRR识别方式和抗原(样)受体识别方式两类,并绘出危险因子识别方式分类汇总图。综合危险因子的属性、分类和生命机体对危险因子的识别方式,绘制出危险因子与生命机体机制信息的相关互动图,从保守性结构信息、生理产物信息、对机体损伤信息、代谢通路信息、对机体变应信息、精细纯外观构象信息及内在性核心信息7个方面对两者的相互关系进行了引证和分析,进一步阐释了生命信息安全控制机制。     

Membranous structures transfer cell surface proteins across NK cell immune synapses

Intercellular transfer of cell surface proteins is widespread and facilitates several recently discovered means for immune cell communication. Here, we examined the molecular mechanism for intercellular exchange of the natural killer (NK) cell receptor KIR2DL1 and HLA-C, prototypical proteins that swap between NK cells and target cells. Transfer was contact dependent and enhanced for cells expressing cognate receptor/ligand pairs but did not depend on KIR2DL1 signaling. To a lesser extent, proteins transferred independent from specific recognition. Intracellular domains of transferred proteins were not exposed to the extracellular environment and transferred proteins were removed by brief exposure to low pH. By fluorescence microscopy, transferred proteins localized to discrete regions on the recipient cell surface. Higher resolution scanning electron micrographs revealed that transferred proteins were located within specific membranous structures. Transmission electron microscopy of the immune synapse revealed that membrane protrusions from one cell interacted with the apposing cell surface within the synaptic cleft. These data, coupled with previous observations, lead us to propose that intercellular protein transfer is mediated by membrane protrusions within and surrounding the immunological synapse.