Helical Ends Research Articles

Tailoring helical ends of π-extended [6]heterohelicenes to control optical, and electrochemical features.

The inherent helical chirality and improved π-stacking capabilities endow helicenes with fascinating photophysical characteristics when decorated with lateral π-extensions. Here, we report the synthesis and physicochemical characterization of expanded hetero[6]helicenes fused with thiadiazole and selenadiazole rings at the helical ends. Comparing these heterohelicenes revealed the impact of the heteroatom-embedded aromatic rings on the excited state and redox features. A small structural variation of the terminal rings from thiadiazole to selenadiazole caused a striking change in the heterohelical nanographenes.

Artificial Water Channels—Progress Innovations and Prospects

Artificial Water Channels—Progress Innovations and Prospects

Controlling the Emissive, Chiroptical, and Electrochemical Properties of Double [7] Helicenes through Embedded Aromatic Rings.

We present here the synthesis and in-depth physicochemical characterization of a double hetero[7]helicene fused with four triazole rings at both helical ends. The comparison of this triazole-fused double helicene with the previously reported all-carbon and thiadiazole-fused analogs revealed the huge impact of the embedded aromatic rings on the photophysical features. The small structural variation of the terminal rings from thiadiazole to triazole caused a dramatic change of the photoluminescence quantum yields (PLQYs) from <1 % to 96 %, while the replacement of the terminal benzene rings with triazole rings induced a tenfold enhancement of the circularly polarized luminescence dissymmetry factor. These observations were well corroborated with transient absorption analysis and/or theoretic calculations. In addition, the triazole-fused double helicene exhibited ambipolar redox behavior, enabling the generation of radical cation and anion species by electrochemical and chemical methods and showing its potential for spin-related applications.

Oxygen diffusion pathways in mutated forms of a LOV photoreceptor from Methylobacterium radiotolerans: A molecular dynamics study.

Mr4511 from Methylobacterium radiotolerans is a photoreceptor of the light, oxygen voltage (LOV) family, binding flavin mononucleotide (FMN) as a chromophore. It exhibits the prototypical LOV photocycle, with the reversible formation of an FMN-Cys71 adduct via fast decay of the FMN triplet state. Mr4511 has high potential as a photosensitiser for singlet oxygen (SO) upon mutation of C71. Mr4511-C71S shows a triplet lifetime (τ T) of several hundreds of microseconds, ensuring efficient energy transfer to dioxygen to form SO. In this work, we have explored the potential diffusion pathways for dioxygen within Mr4511 using molecular dynamics (MD) simulations. The structural model of wild-type (wt) Mr4511 showed a dimeric structure stabilised by a strong leucine zipper at the two C-terminal helical ends. We then introduced in silico the C71S mutation and analysed transient and persistent oxygen channels. MD simulations indicate that the chromophore binding site is highly accessible to dioxygen. Mutations that might favour SO generation were designed based on their position with respect to FMN and the oxygen channels. In particular, the C71S-Y61T and C71S-Y61S variants showed an increased diffusion and persistence of oxygen molecules inside the binding cavity.

Furan-containing double tetraoxa[7]helicene and its radical cation.

An unprecedented furan-based double oxa[7]helicene 1 was achieved, featuring a stable twisted conformation with π-overlap at both helical ends. The excellent conformational stability allowed for optical resolution of 1, which provided a pair of enantiomers exhibiting pronounced mirror-imaged circular dichroism and circularly polarized luminescence activity. The radical cation of 1 was obtained by chemical oxidation as evidenced by UV-Vis-NIR absorption, electron paramagnetic resonance spectroscopy and in situ spectroelectrochemistry. The present work is the starting point for the investigation of open-shell oxahelicenes.

Exploring the Transport Mechanism of the Human AE4 (SLC4A9) CL-/HCO3- Exchanger

The SLC4 family includes Na+-independent Cl-/HCO3- exchangers and Na+-coupled HCO3- transporters. SLC4 proteins are crucial for important physiological processes, including CO2 transport, intracellular and extracellular pH regulation and HCO3- transport in epithelia. The AE4 (SLC4A9) protein plays a role in NaCl reabsorption and Cl--dependent fluid secretion in distal nephron and salivary glands respectively; however, its transport mechanism remains controversial. It has been proposed that AE4 mediates Cl-/HCO3- exchange, Na+-HCO3- cotransport or Cl-/cation-HCO3- exchange suggesting that shares functional features with both the Cl-/HCO3- exchanger AE1(SLC4A1) and the Na+-HCO3- co-transporter NBCe1 (SLC4A4). Our sequence alignments show that AE1, NBCe1 and AE4 share high amino-acidic identity in the transmembrane domain. Functionally relevant residues in AE1 and NBCe1 are conserved in AE4, suggesting that the ion transport mechanism should be encoded by the same set of residues. We explored the transport mechanism of the human version of AE4 using molecular modeling, site-directed mutagenesis and functional assays. Our molecular simulations show that the proposed anion-binding site in AE1, between the helical ends of TM 3 and TM10, is also present in AE4. Three residues at this region have been postulated to be crucial for anion transport in AE1 and NBCe1. We mutated homologous D709, T448 and I758 in AE4 and found that only T448 is functionally important. Additional mutations of S754 and T756 in this region dońt show functional changes compared to WT AE4. These results suggest that the predicted ion-binding site in AE4 requires T448 to support the transport cycle but, in contrast to AE1 and NBCe1, residues at equivalent positions 709 and 758 are not important for function.

Preferential Rotation of Chiral Dipoles in Isotropic Turbulence.

We introduce a new particle shape which shows preferential rotation in three dimensional homogeneous isotropic turbulence. We call these particles chiral dipoles because they consist of a rod with two helices of opposite handedness, one at each end. 3D printing is used to fabricate these particles with a length in the inertial range and their rotations are tracked in a turbulent flow between oscillating grids. High aspect ratio chiral dipoles preferentially align with their long axis along the extensional eigenvectors of the strain rate tensor, and the helical ends respond to the extensional strain rate with a mean spinning rate that is nonzero. We use Stokesian dynamics simulations of chiral dipoles in pure strain flow to quantify the dependence of spinning on particle shape. Based on the known response to pure strain, we build a model that gives the spinning rate of small chiral dipoles using velocity gradients along Lagrangian trajectories from high resolution direct numerical simulations. The statistics of chiral dipole spinning determined with this model show surprisingly good agreement with the measured spinning of much larger chiral dipoles in the experiments.

"Sticky"-Ends-Guided Creation of Functional Hollow Nanopores for Guest Encapsulation and Water Transport.

Commercial uses of water-transporting aquaporins for seawater desalination and wastewater reclamation/reuse are being investigated in both academia and the industry. Presently, structural complexity, stability, scalability, and activity reconstitution of these costly channel proteins still present substantial challenges to scientists and engineers. An attractive strategy is to develop robust synthetic water channels able to mimic the water-transporting function of aquaporins for utility in the making of next generation of water channel-based biomimetic porous membranes for various water purification applications. In sharp contrast to burgeoning development in constructing synthetic ion channels over the past four decades, very limited progress has been made in the area of synthetic water channels. A handful of such examples include the first report by Percec in 2007 (Percec et al. J. Am. Chem. Soc. 2007, 129, 11698-11699), which was followed by Barboiu in 2011 (Barboiu et al. Angew. Chem., Int. Ed. 2011, 50, 11366-11372), Gong and Hou in 2012 (Gong et al. Nat. Commun. 2012, 3, 949; Hou et al. J. Am. Chem. Soc. 2012, 134, 8384-8387), and Zeng in 2014 (Zeng et al. J. Am. Chem. Soc. 2014, 136, 14270-14276). Radically deviating from the fact that the discovery of novel synthetic channel systems with desired transport selectivity is most often empirical and very often serendipitous, we have instead adopted a more rational designer approach whereby molecular building blocks have been carefully designed from scratch to perform their intended built-in functions. Our designer journey started in 2008, two years after I started leading a group at the National University of Singapore. Since then, we have been actively investigating the use of designed water-binding "aquafoldamers" to construct synthetic water channels for the rapid and selective transport of water molecules ideally with the exclusion of all other nonproton molecular species. Toward this goal, we designed and characterized, by an experimental-theoretical synergy, a new class of modular, H-bonded, and crescent-shaped oligopyridine amide foldamers, enclosing a sizable cavity of about 2.8 Å in diameter. Matching well with the diameter of water molecules and decorated by interior-pointing H-bond donors (amide H atoms) and acceptors (pyridine N atoms) for water binding, this sizable cavity experimentally proves to be suitable for water recognition. In particular, helically folded oligomers are found to be capable of binding two water molecules that are vertically aligned in parallel with helical axis. However, the existence of two repulsive groups at the two helical ends prevents the formation of 1D hollow tubular cavity, via self-assembly, for encapsulating 1D water chains. Subsequently, we introduced two electrostatically complementary functional groups that act as "sticky" ends at helical ends. These feeble "sticky" ends faithfully and seamlessly align short cavity-containing helices one-dimensionally to create hollow tubular aquapores. To our delight, these aquapores demonstrate their excellent ability of highly selectively hosting a chain of single file H-bonded water molecules and allow for selective transport of both protons and water molecules with exclusion of metal ions including Na(+) and K(+) ions across the lipid membranes.

Conformational Parameters for Amino Acids in Helical, β-Sheet, and Random Coil Regions Calculated from Proteins: After 40 Years

Forty years ago, Peter Y. Chou and Gerald D. Fasman (1974a), relying on the information from fifteen proteins calculated α helix, β-sheet, and coil conformational parameters, Pα,Pβ,and Pc,for the 20 naturally occurring amino acids from the frequency of occurrence of each amino acid residue in the α, β, and coil conformations. Secondary structure of these 15 proteins had been determined by X-ray crystallography. Although the accuracy could not go over to the level of 60% too much, these values utilized for a long time to provide a simple procedure, devoid of complex computer calculations, to predict the secondary structure of proteins from their known amino acid sequences. In the same article of Peter Y. Chou and Gerald D. Fasman, a detailed analysis of the helix and β-sheet boundary residues in proteins provided amino acid frequencies at the N-and C-terminal ends which were used to delineate helical and β regions. Charged residues are found with the greatest frequency at both helical ends, but they were mostly absent in β-sheet regions. In the same article a mechanism of protein folding was proposed, whereby helix nucleation starts at the centers of the helix where the Pα values are highest, and propagates in both directions, until strong helix breakers where Pα values are lowest, terminate the growth at both ends. Similarly, residues with the highest Pβ values will initiate β regions and residues with the lowest Pβ values will terminate β regions. The helical region with the largest Pα was proposed as the site of the first fold during protein renaturation. The mechanism whereby proteins fold into their native conformation, capable of biological activity, has been a long sought after goal. With the elucidation of the three-dimensional structure of many proteins through X-ray crystallography, a new momentum has been given to understanding the factors governing this complex assembly of polypeptide chains. In this paper, using similar statistics from 20 347 proteins, the level of reliability of formerly found results is discussed.

Rational Design of Multilayer Collagen Nanosheets with Compositional and Structural Control.

Two collagen-mimetic peptides, CP(+) and CP(-), are reported in which the sequences comprise a multiblock architecture having positively charged N-terminal (Pro-Arg-Gly)3 and negatively charged C-terminal (Glu-Hyp-Gly)3 triad extensions, respectively. CP(+) rapidly self-associates into positively charged nanosheets based on a monolayer structure. In contrast, CP(-) self-assembles to form negatively charged monolayer nanosheets at a much slower rate, which can be accelerated in the presence of calcium(II) ion. A 2:1 mixture of unassociated CP(-) peptide with preformed CP(+) nanosheets generates structurally defined triple-layer nanosheets in which two CP(-) monolayers have formed on the identical surfaces of the CP(+) nanosheet template. Experimental data from electrostatic force microscopy (EFM) image analysis, zeta potential measurements, and charged nanoparticle binding assays support a negative surface charge state for the triple-layer nanosheets, which is the reverse of the positive surface charge state observed for the CP(+) monolayer nanosheets. The electrostatic complementarity between the CP(+) and CP(-) triple helical cohesive ends at the layer interfaces promotes a (CP(-)/CP(+)/CP(-)) compositional gradient along the z-direction of the nanosheet. This structurally informed approach represents an attractive strategy for the fabrication of two-dimensional nanostructures with compositional control.

Development of a Slow-Switching Dronpa Variant for 2-Color Super Resolution Imaging of Drp1 During Mitochondrial Fission

We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein (PS-FP) and a popular marker for photo-activated localization microscopy (PALM). We found the excitation light photo-activates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a novel Dronpa variant, rsKame. The increased steric hindrance generated by the mutation reduced the excitation light-induced photo-conversion from the dark to fluorescent state. To demonstrate the applicability of rsKame, we paired it with PAmCherry1 in a 2-color PALM imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We quantified Drp1 helical ring diameters and lengths showing that the rings undergo changes in diameter between different stages of mitochondrial fission without significant modification of their length. These results support the twistase model of Drp1 constriction with some loss of subunits at the helical ends.

Low-temperature molecular dynamics simulations of horse heart cytochrome c and comparison with inelastic neutron scattering data

Molecular dynamics (MD) simulation combined with inelastic neutron scattering can provide information about the thermal dynamics of proteins, especially the low-frequency vibrational modes responsible for large movement of some parts of protein molecules. We performed several 30-ns MD simulations of cytochrome c (Cyt c) in a water box for temperatures ranging from 110 to 300 K and compared the results with those from experimental inelastic neutron scattering. The low-frequency vibrational modes were obtained via dynamic structure factors, S(Q, ω), obtained both from inelastic neutron scattering experiments and calculated from MD simulations for Cyt c in the same range of temperatures. The well known thermal transition in structural movements of Cyt c is clearly seen in MD simulations; it is, however, confined to unstructured fragments of loops Ω1 and Ω2; movement of structured loop Ω3 and both helical ends of the protein is resistant to thermal disturbance. Calculated and experimental S(Q, ω) plots are in qualitative agreement for low temperatures whereas above 200 K a boson peak vanishes from the calculated plots. This may be a result of loss of crystal structure by the protein-water system compared with the protein crystal.

Crystal Structure of the N-terminal Domain of the Yeast General Corepressor Tup1p and Its Functional Implications

The yeast Cyc8p-Tup1p protein complex is a general transcriptional corepressor of genes involved in many different physiological processes. Herein, we present the crystal structure of the Tup1p N-terminal domain (residues 1-92), essential for Tup1p self-assembly and interaction with Cyc8p. This domain tetramerizes to form a novel antiparallel four-helix bundle. Coiled coil interactions near the helical ends hold each dimer together, whereas interdimeric association involves only two sets of two residues located toward the chain centers. A mutagenesis study confirmed that the nonpolar residues responsible for the association of the protomers as dimers are also required for transcriptional repression. An additional structural study demonstrated that the domain containing an Leu(62) → Arg mutation that had been shown not to bind Cyc8p exhibits an altered structure, distinct from the wild type. This altered structure explains why the mutant cannot bind Cyc8p. The data presented herein highlight the importance of the architecture of the Tup1p N-terminal domain for self-association.

Secondary and Tertiary Structure of Bacteriorhodopsin in the SDS Denatured State

We characterized the structure of partially unfolded bacteriorhodopsin in sodium dodecyl sulfate (SDS) micelles and compared it with its in vitro refolded structure after reconstitution with dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (DMPC/CHAPS). Intrahelical and interhelical distances were mapped in the protein using strategically located spin-label pairs at helical ends, assayed by pulsed electron paramagnetic resonance spectroscopy (double electron-electron spin resonance, DEER). We find that in SDS the intrahelical end-to-end distances exhibit broad distributions, suggesting a heterogeneous ensemble of conformations with differing secondary structures. Nevertheless, a majority of the denatured population retains end-to-end distances similar to those in the native state. In contrast, the observed greatly increased interhelical distances, in addition to their very broad distributions, suggest that in the SDS micelles very little of the native tertiary structure remains.

Differential structure of the intronic promoter of the Bombyx mori A3 actin gene correlated with silkworm sensitivity/resistance to nucleopolyhedrovirus

Previous reports demonstrated that actin is necessary for nucleocapsid transport and viral gene expression during nucleopolyhedrovirus infection of Bombyx mori. The first intron of B. mori A3 actin contains a cryptic promoter that drives expression of a rare isoform. We detected differences in the size and nucleotide composition of the first intron of the A3 actin gene from B. mori strain C24A, which is more resistant to nucleopolyhedrovirus than the M11A strain (22 and 95% lethality, respectively). We sought to determine if resistance to BmMNPV infection and the A3 actin promoter structure are correlated. Intrinsically bent DNA sites in these sequences, which determine curved structures, were analyzed by electrophoretic mobility assays and the helical parameters ENDS ratio, roll and twist. We found both fragments to have non-centralized bent DNA sites with distinct ENDS ratio values, nucleotide positions and two-dimensional structures. Additionally, a conformational-sensitive gel electrophoresis assay identified an allelic variation found in strain M11A that is absent in strain C24A. These data suggest that A3 actin intronic sequence variations impair virus propagation and are markers of BmMNPV-resistant populations.

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00 degrees values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

Tissue-specific Changes in the Hydroxylysine Content and Cross-links of Collagens and Alterations in Fibril Morphology in Lysyl Hydroxylase 1 Knock-out Mice

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.

Examining the Influence of Linkers and Tertiary Structure in the Forced Unfolding of Multiple-Repeat Spectrin Molecules

The unfolding pathways of multiple-repeat spectrin molecules were examined using steered molecular dynamics (SMD) simulations to forcibly unfold double- and triple-repeat spectrin molecules. Although SMD has previously been used to study other repeating-domain proteins, spectrin offers a unique challenge in that the linker connecting repeat units has a definite secondary structure, that of an α-helix. Therefore, the boundary conditions imposed on a double- or triple-repeat spectrin must be carefully considered if any relationship to the real system is to be deduced. This was accomplished by imposing additional forces on the system which ensure that the terminal α-helices behave as if there were no free noncontiguous helical ends. The results of the SMD simulations highlight the importance of the rupture of the α-helical linker on the subsequent unfolding events. Rupture of the linker propagates unfolding in the adjacent repeat units by destabilizing the tertiary structure, ultimately resulting in complete unfolding of the affected repeat unit. Two dominant classes of unfolding pathways are observed after the initial rupture of a linker which involve either rupture of another linker (possibly adjacent) or rupture of the basic tertiary structure of a repeat unit. The relationship between the force response observed on simulation timescales and those of experiment or physiological conditions is also discussed.

The Fusion Activity of HIV-1 gp41 Depends on Interhelical Interactions

Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function.

無研削圧縮コイルばね調査報告

Usually, helical compression spring ends require a grinding process. This process sometimes restricts productivity of the production line and needs countermeasures for dust and noise. Fine steel powder generated from the process contains particles of the abrasive wheel. As the steel powder and the abrasive particles are inseparably mixed, they cannot be handled as steel material for scrap metal. They inevitably become industrial waste. As a result, the grinding process needs additional costs for dust control, noise prevention and waste disposal. This committee was launched for the purpose of investigating the new processing method which can replace grinding operation, and the following results were obtained.(1) Study on current practicesSprings that have not-ground- ends are widely used for industrial purposes. Wire diameter of the springs range from 0.2mm to 35mm and the spring indexes are higher than 3.5. The production-mix ratio between “ground-ends springs” and “not-ground-ends” springs varies from company to company. It seems that the customers make a decision on necessity of coil-end grinding. When squareness and flatness is essential, the grinding process is employed.(2) Archive researchThe committee members reviewed relevant literatures written from 1975 to August 2003 and could not find any that conforms to their purposes.(3) Patent researchThe committee searched through patents applied in the same period and found that 10 of them met with the requirements.(4) Finite Element Analysis (FEA)The committee investigated the applicable range of a spring characteristic formula and a stress formula by applying FEA to the calculation model. Consequently, it seems that the spring characteristic formula is practical. However, when the spring index became less than six, it is necessary to take the influence of contact stress into consideration for the stress calculation formula.(5) Study on existing manufacturing methodsBending, rolling or cutting-off method, are mainly used for the manufacturing of “not-ground-ends springs.” The Rolling method has long been utilized for hot-formed springs while it has not been applied to cold-formed springs.