Plasmonic Resonance Excited Extinction Spectra of Cross-Shaped Ag Nanoparticles

Plasmonic properties of cross-shaped Ag nanoparticles are investigated theoretically using finite-difference time-domain algorithm. Electric field (E-field) distribution of a single cross-shaped Ag nanoparticle with different shape parameters and patterned nanoparticles with different periods were presented. Both red shift and blue shift of the extinction spectra were observed. The simulation results demonstrated that the strong E-field intensity is located at sharp corner of the nanoparticles. And E-field intensity of the nanoparticle array is much stronger than that of a single Ag nanoparticle. Enhancement of the large localized E-field originating from the nanoparticles was analyzed. Corresponding influence of “hot spots” effect on enhancing Raman scattering was discussed as well.     

Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering

Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.     

Numerical Study of an Ultra-Broadband and Polarization Independence Metamaterial Cross-Shaped Fractal Absorber

A three-dimensional cross-shaped fractal metamaterial absorber with ultra-wide wavelength band, polarization-independence and wide-angle, is numerically investigated by the finite-difference time-domain method. In this absorber, the solar energy is trapped by the cross-shaped fractal of the upper layer, and the Si-ring filled with iron in the middle layer and the wavelength band can be broadened by the self-similarity of fractal structure. The absorber exhibits absorptivity higher than 91% for the wavelengths from 400 to 2000 nm and an absorption bandwidth of about 133%. Furthermore, the proposed absorber realizes polarization independence, and the maximum incident angle is 76°. However, as the iron material applied in the nano-metamaterial absorber (NMA) can be easily oxidized and rusted, it is replaced by nickel with characteristics such as corrosion resistance and high-temperature resistance; thus, an improved NMA is obtained. The improved absorber not only eliminates the corrosion-prone defects of the above proposed structure but also maintains polarization independence and high absorption and widens the angle of incidence up to 79° and thereby can be applied in many areas, such as solar energy harvesting.

     

Structure of a mouse immunoglobulin G that lacks the entire CH1 domain: protein sequencing and small-angle X-ray scattering studies

The structure of a short-chain IgG2a antibody, which is a member of the family of mouse anti-dansyl switch variant antibodies with identical variable regions but different heavy-chain constant regions [Dangl, J.L., Parks, D. R., Oi, V. T., & Herzenberg, L. A. (1982) Cytometry 2, 395-401], is reported. Amino acid sequencing analyses have demonstrated that in the short-chain IgG2a antibody the entire CH1 domain is deleted whereas the hinge region remains intact. Small-angle X-ray scattering data were collected for the short-chain IgG2a antibody and compared with those for the switch variant IgG1, IgG2a, and IgG2b antibodies with the normal heavy chain. It has been concluded that deletion of the CH1 domain results in a large structural change and the short-chain IgG2a antibody possesses an elongated molecular shape with a much smaller hinge angle as compared with the normal IgG2a antibody that is a Y-shaped molecule.     

Determinants of human glucokinase activation and implications for small molecule allosteric control

Glucokinase (GK) is an enzyme that catalyzes the ATP-dependent phosphorylation of glucose to form glucose-6-phosphate, and it is a tightly regulated checkpoint in glucose homeostasis. GK is known to undergo substantial conformational changes upon glucose binding. The monomeric enzyme possesses a highly exotic kinetic activity profile with an unusual sigmoidal dependence on glucose concentration. In this interdisciplinary study, which draws on small angle X-ray scattering (SAXS) integrated with 250?ns of atomistic molecular dynamics (MD) simulations and experimental glucose binding thermodynamics, we reveal that the critical regulation of this glucose sensor is due to a solvent controlled “switch”. We demonstrate that the “solvent switch” is driven by specific protein structural dynamics, which leads to an enzyme structure that has a much more favorable solvation energy than most of the protein ensemble. These findings uncover the physical workings of an agile and flexible protein scaffold, which derives its long-range allosteric control through specific regions with favorable solvation energy. The physiological framework presented herein provides insights that have direct implications for the design of small molecule GK activators as anti-diabetes therapeutics as well as for understanding how proteins can be designed to have built-in regulatory functions via solvation energy dynamics.     

A reinterpretation of neutron scattering experiments on a lipidated Ras peptide using replica exchange molecular dynamics

The Ras family of proteins plays crucial roles in a variety of cell signaling networks where they have the function of a molecular switch. Their particular medical relevance arises from mutations in these proteins that are implicated in ~30% of human cancers. The various Ras proteins exhibit a high degree of homology in their soluble domains but extremely high variability in the membrane anchoring regions that are crucial for protein function and are the focus of this study. We have employed replica exchange molecular dynamics computer simulations to study a doubly lipidated heptapeptide, corresponding to the C-terminus of the human N-Ras protein, incorporated into a dimyristoylphosphatidylcholine lipid bilayer. This same system has previously been investigated experimentally utilizing a number of techniques, including neutron scattering. Here we present results of well converged simulations that describe the subtle changes in scattering density in terms of the location of the peptide and its lipid modifications and in terms of changes in phospholipid density arising from the incorporation of the peptide into the membrane bilayer. The detailed picture that emerges from the combination of experimental and computational data exemplifies the power of combining isotopic substitution neutron scattering with atomistic molecular dynamics simulation. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Laminin-nidogen complex. Extraction with chelating agents and structural characterization

Large quantities of intact laminin-nidogen complex could be extracted from a mouse tumor basement membrane with a physiological buffer containing EDTA. Analysis of the purified complex demonstrated that the two proteins occur in an equimolar ratio and that anchoring of these complexes to the extracellular matrix requires divalent cations. Reversible dissociation of the complex was achieved with 2 M guanidine X HCl and has been used for purification of the individual components. Electron microscopy and binding studies using laminin fragments demonstrated that nidogen interacts specifically with the center of the cross-shaped laminin molecule as represented by the short-arm structure fragment 1. The complex was also useful to confirm and refine a previously proposed dumb-bell structure of nidogen and to prepare and characterize the cell-binding fragment 8 from the long arm of laminin.     

Fractal dimension of antibody‐PEG precipitate: Light microscopy for the reconstruction of 3D precipitate structures

Protein and in particular antibody precipitation by PEG is a cost‐effective alternative for the first capture step. The 3D structure of precipitates has a large impact on the process parameters for the recovery and dissolution, but current technologies for determination of precipitate structures are either very time consuming (cryo‐TEM) or only generate an average fractal dimension (light scattering). We developed a light microscopy based reconstruction of 3D structures of individual particles with a resolution of 0.1–0.2 µm and used this method to characterize particle populations generated by batch as well as continuous precipitation in different shear stress environments. The resulting precipitate structures show a broad distribution in terms of fractal dimension. While the average fractal dimension is significantly different for batch and continuous precipitation, the distribution is broad and samples overlap significantly. The precipitate flocs were monofractal from micro‐ to nanoscale showing a random but consistent nature of precipitate formation. We showed that the fractal dimension and 3D reconstruction is a valuable tool for characterization of protein precipitate processes. The current switch from batch to continuous manufacturing has to take the 3D structure and population of different protein precipitates into account in their design, engineering, and scale up.     

Identification of molecules in leech extracellular matrix that promote neurite outgrowth

The molecular composition of the substrate is of critical importance for neurite extension by isolated identified leech nerve cells in culture. One substrate upon which rapid growth occurs in defined medium is a cell-free extract of extracellular matrix (ECM) that surrounds the leech central nervous system (CNS). Here we report the co-purification of neurite-promoting activity with a laminin-like molecule. High molecular mass proteins from leech ECM purified by gel filtration exhibited increased specific activity for promoting neurite outgrowth. The most active fractions contained three major polypeptide bands of ca. 340, 250 and 220 kDa. Electron microscopy of rotary-shadowed samples showed three macromolecules, one of which had a cross-shaped structure similar to vertebrate laminin. A second six-armed molecule resembled vertebrate tenascin and a third rod-like molecule resembled vertebrate collagen type IV. The most active fractions contained a protein of ca. 1 MDa on non-reducing gels with disulphide-linked subunits of ca. 220 and 340 kDa, with cross-shaped laminin-like molecules. We conclude that a laminin-like molecule represents a major neurite promoting component present in leech ECM. The experiments represent a first step in determining the location of leech laminin within the CNS and assessing its role in neurite outgrowth during development and regeneration.     

Three-dimensional structure of the KChIP1-Kv4.3 T1 complex reveals a cross-shaped octamer

Brain I(A) and cardiac I(to) currents arise from complexes containing Kv4 voltage-gated potassium channels and cytoplasmic calcium-sensor proteins (KChIPs). Here, we present X-ray crystallographic and small-angle X-ray scattering data that show that the KChIP1-Kv4.3 N-terminal cytoplasmic domain complex is a cross-shaped octamer bearing two principal interaction sites. Site 1 comprises interactions between a unique Kv4 channel N-terminal hydrophobic segment and a hydrophobic pocket formed by displacement of the KChIP H10 helix. Site 2 comprises interactions between a T1 assembly domain loop and the KChIP H2 helix. Functional and biochemical studies indicate that site 1 influences channel trafficking, whereas site 2 affects channel gating, and that calcium binding is intimately linked to KChIP folding and complex formation. Together, the data resolve how Kv4 channels and KChIPs interact and provide a framework for understanding how KChIPs modulate Kv4 function.     

HIV-1 capsid protein forms spherical (immature-like) and tubular (mature-like) particles in vitro: structure switching by pH-induced conformational changes.

The viral genome and replicative enzymes of the human immunodeficiency virus are encased in a shell consisting of assembled mature capsid protein (CA). The core shell is a stable, effective protective barrier, but is also poised for dissolution on cue to allow transmission of the viral genome into its new host. In this study, static light scattering (SLS) and dynamic light scattering (DLS) were used to examine the entire range of the CA protein response to an environmental cue (pH). The CA protein assembled tubular structures as previously reported but also was capable of assembling spheres, depending on the pH of the protein solution. The switch from formation of one to the other occurred within a very narrow physiological pH range (i.e., pH 7.0 to pH 6.8). Below this range, only dimers were detected. Above this range, the previously described tubular structures were detected. The ability of the CA protein to form a spherical structure that is detectable by DLS but not by electron microscopy indicates that some assemblages are inherently sensitive to perturbation. The dimers in equilibrium with these assemblages exhibited distinct conformations: Dimers in equilibrium with the spherical form exhibited a compact conformation. Dimers in equilibrium with the rod-like form had an extended conformation. Thus, the CA protein possesses the inherent ability to form metastable structures, the morphology of which is regulated by an environmentally-sensitive molecular switch. Such metastable structures may exist as transient intermediates during the assembly and/or disassembly of the virus core.     

Solution structure of a short DNA fragment studied by neutron scattering

The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small-angle scattering. In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B-DNA. The neutron scattering curve is well fitted by that of a rigid rod with a length of 44 nm and a diameter of 2 nm. The results were confirmed by quasi-elastic light scattering and analytical centrifugation. The neutron measurements in H2O and D2O buffer reveal a cross-sectional inhomogeneity not detected by X-ray small-angle scattering. This inhomogeneity is caused by the hydration layer around the DNA core and not by the helical structure. The primary solvent shell has a density increased by at least 4-9% compared to bulk water.     

Heterosynapsis in a heterozygous fertile boar carrier of a 3;7 translocation

Silver-stained synaptonemal complexes (SCs) in surface-spread pachytene nuclei from a boar, heterozygous for a reciprocal translocation, were analysed by electron microscopy. In such heterozygotes, cross-shaped quadrivalent configurations are expected to form in order to maximize homologous pairing. Contrary to the classical, expected cross-shaped configuration, heterosynapsis was often observed, with asymmetrical association in the lateral elements of the non-homologous partners of the quadrivalents. This heterosynapsis is assumed to be a mechanism preventing spermatocyte loss, but inducing a secondary segregational type of impairment of fertility due to foetal wastage leading to reduced prolificacy.     

Terahertz Dual-Band Polarization Insensitive Electromagnetically Induced Transparency-Like Metamaterials

In this paper, an electromagnetically induced transparency-like metamaterial for terahertz is designed. The structure is based on cross-shaped and SRRs composite elements. It can achieve dual-frequency transparent window in a wide frequency band and is insensitive to electromagnetic wave polarization. The resonance points of transmission peaks are 198.55 and 254.18 GHz, respectively. The electromagnetic transmittance can reach 95.6% and 97.7%, respectively, which has excellent electromagnetic transmission effect. The measured results are in good agreement with the trend of simulation curve. At the same time, flexible polyimide with stable performance is selected as the base material of metamaterial dielectric, which can be widely used in microwave fields such as filters, sensors, and slow light devices.

     

Three-dimensional structure of the surface protein of Desulfurococcus mobilis.

The spherical cells of the thermophilic, sulfur-dependent archaebacterium Desulfurococcus mobilis are completely covered with a relatively poorly ordered, tetragonally arrayed surface protein. The structure of this surface protein was examined by using three-dimensional electron microscopy. The protein lattice forms an open meshwork composed of cross-shaped morphological units, which are released when glycerol is added. These subunits make contact at the distal ends of their four arms. The p4 symmetry requires that each of these morphological subunits represents a tetramer. The strong interaction of the monomers within the crosses and the relatively weak interaction of the intersecting arms of the crosses within the lattice structure suggest that the tetramers are assembled before their incorporation into the lattice.     

Structural mechanics of the pH-dependent activity of beta-carbonic anhydrase from Mycobacterium tuberculosis

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. Carbonic anhydrases are divided into at least three classes and are believed to share a zinc-hydroxide mechanism for carbon dioxide hydration. beta-carbonic anhydrases are broadly spread among the domains of life, and existing structures from different organisms show two distinct active site setups, one with three protein coordinations to the zinc (accessible) and the other with four (blocked). The latter is believed to be inconsistent with the zinc-hydroxide mechanism. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta-carbonic anhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. We have recently solved the structure of this protein, which was a dimeric protein with a blocked active site. Here we present the structure of the thiocyanate complexed protein in a different crystal form. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta-carbonic anhydrase can switch between the two states. A pH-dependent dimer to tetramer equilibrium was also demonstrated by dynamic light scattering measurements. The data presented here, therefore, suggest a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.     

The crystal structure of Aq_328 from the hyperthermophilic bacteria Aquifex aeolicus shows an ancestral histone fold

The structure of Aq_328, an uncharacterized protein from hyperthermophilic bacteria Aquifex aeolicus, has been determined to 1.9 A by using multi-wavelength anomalous diffraction (MAD) phasing. Although the amino acid sequence analysis shows that Aq_328 has no significant similarity to proteins with a known structure and function, the structure comparison by using the Dali server reveals that it: (1) assumes a histone-like fold, and (2) is similar to an ancestral nuclear histone protein (PDB code 1F1E) with z-score 8.1 and RMSD 3.6 A over 124 residues. A sedimentation equilibrium experiment indicates that Aq_328 is a monomer in solution, with an average sedimentation coefficient of 2.4 and an apparent molecular weight of about 20 kDa. The overall architecture of Aq_328 consists of two noncanonical histone domains in tandem repeat within a single chain, and is similar to eukaryotic heterodimer (H2A/H2B and H3/H4) and an archaeal histone heterodimer (HMfA/HMfB). The sequence comparisons between the two histone domains of Aq_328 and six eukaryotic/archaeal histones demonstrate that most of the conserved residues that underlie the Aq_328 architecture are used to build and stabilize the two cross-shaped antiparallel histone domains. The high percentage of salt bridges in the structure could be a factor in the protein's thermostability. The structural similarities to other histone-like proteins, molecular properties, and potential function of Aq_328 are discussed in this paper.     

Co-translational myristoylation alters the quaternary structure of HIV-1 Nef in solution

We have studied the solution properties of Nef, a 24-kDa cotranslationally myristoylated protein produced by HIV-1 and other primate lentiviruses. Nef is found in the cytosol and also in association with cytoplasmic membranes, the latter, mediated in part by the myristoyl group attached to the N-terminal glycine. Recombinant Nef was coexpressed in Escherichia coli in tandem with N-myristoyl-transferase and is fully myristoylated. Analysis by circular dichroism showed the myristoylated form to contain a greater alpha-helical content than the nonmyristoylated form. Analysis of modified and unmodified Nef in solution using small angle X-ray scattering, dynamic laser light scattering and analytical ultracentrifugation consistently showed differences in the oligomeric states of the two forms of Nef. Myristoylated Nef is predominantly monomeric and small oligomers which are also present, can be converted to the monomeric form under reducing conditions. By contrast, the nonmyristoylated form exists as a stable hexadecamer in solution which disassociates into tetramers upon addition of reducing agents. Shape reconstructions from small angle scattering curves of nonmyristoylated Nef are compatible with a large disc-like structure in the hexadecameric oligomer consisting of four Nef tetramers. From these findings, we hypothesize that Nef undergoes a substantial conformational change from an "open" into a "closed" form whereby the myristate group is sequestered in a hydrophobic pocket. The myristoylated protein can switch to the open conformation by association of the N-terminal region of molecule with membranes. These changes would allow Nef to carry out various functions depending on the conformational and oligomeric states.     

Structural and Functional Analysis of BipA,a Regulator of Virulence in Enteropathogenic Escherichia coli

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3′, 5′-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes to small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. This molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.     

Mechanisms of isoform-specific residue influence on GTP-bound HRas,KRas, and NRas

HRas, KRas, and NRas are GTPases with a common set of effectors that control many cell-signaling pathways, including proliferation through Raf kinase. Their G-domains are nearly identical in sequence, with a few isoform-specific residues that have an effect on dynamics and biochemical properties. Here, we use accelerated molecular dynamics (aMD) simulations consistent with solution x-ray scattering experiments to elucidate mechanisms through which isoform-specific residues associated with each Ras isoform affects functionally important regions connected to the active site. HRas-specific residues cluster in loop 8 to stabilize the nucleotide-binding pocket, while NRas-specific residues on helix 3 directly affect the conformations of switch I and switch II. KRas, the most globally flexible of the isoforms, shows greatest fluctuations in the switch regions enhanced by a KRas-specific residue in loop 7 and a highly dynamic loop 8 region. The analysis of isoform-specific residue effects on Ras proteins is supported by NMR experiments and is consistent with previously published biochemical data.