Coiled Coil in the Stalk Region of ncd Motor Protein Is Nonlocally Sustained

The dimeric structure of kinesin superfamily proteins plays an important role in their motile functions and characteristics. In this study, the coiled-coil-forming property of the stalk region (192−346) of Drosophila ncd, a C-terminal kinesin motor protein, was investigated by synthesizing various peptide fragments. The α helicity of a set of 46-residue peptides spanning the stalk region appeared too low to form a coiled-coil dimer, probably because of insufficient continuity of the hydrophobic residues at (a and d) core positions in amphipathic heptad repeats. On the other hand, several peptides with leucine residues introduced at core positions or with extensional sequences with high α helicity had an advantage in coiled-coil formation. When we analyzed the thermal and urea-induced unfolding of these dimeric peptides, we identified four domains having a relatively high potential to form coiled coils. Among them, three domains on the C-terminal side of the stalk region, i.e., (252−272), (276−330), and (336−346), were in the same heptad frame, although these potential coiled-coil domains were not self-sustaining individually. This is in sharp contrast to the fragment of human kinesin, (332−369), which has an extremely high tendency toward coiled-coil formation. One of the possible triggers for coiled-coil formation of the ncd stalk region may be the interaction between the motor domain and the C-terminal part of the stalk as previously revealed by X-ray crystallography. The residues, S331 and R335, seem to act as a breaking point for α-helix continuity. This would make the region (336−346), as the head−stalk joint, more flexible such as seen with a plus-end-directed kinesin, if this region had no interaction with the motor domain. These characteristic differences between ncd and kinesin suggest that the nonlocally sustained coiled coil of ncd is one of the factors important for minus-end-directed motility

Use of NMR-Based Metabolomics To Chemically Characterize the Roasting Process of Chicory Root

Roasted chicory root (Cichorium intybus) has been widely accepted as the most important coffee substitute. In this study, a nuclear magnetic resonance (NMR)-based comprehensive analysis was performed to monitor the substantial changes in the composition of chicory root during the roasting process. A detailed signal assignment of dried raw and roasted chicory roots was carried out using 1H, 13C, 1H–1H DQF-COSY, 1H–13C edited-HSQC, 1H–13C CT-HMBC, and 1H–13C HSQC-TOCSY NMR spectra. On the basis of the signal assignments, 36 NMR-visible components were monitored simultaneously during roasting. Inulins, sucrose, and most of the amino acids were largely degraded during the roasting process, whereas monosaccharides decreased at the beginning and then increased until the dark roasting stage. Acetamide, 5-hydroxymethylfurfural, di-d-fructose dianhydride, and norfuraneol were newly formed during roasting. Furthermore, a principal component analysis score plot indicated that similar chemical composition profiles could be achieved by roasting the chicory root either at a higher firepower for a shorter time or at a lower firepower for a longer time

Comparison of Peanut Compounds during Roasting and the Effect of Peanut Shells

Peanuts are widely used in a variety of processed foods, and their food properties are attributed to a variety of compounds that change in the roasting process. Here, we investigated the thermal changes in multiple categories of compounds in Virginia-type peanut seeds and peels during roasting using nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–tandem mass spectrometry (LC–MS/MS) as nontargeted analytical methods. Detectable taste and aroma compounds increased up to 2.1- (sucrose), 2.0- (glutamate + pyroglutamate), and 68-fold (4-methyl-5-thiazoleethanol) during roasting. Thermal changes characterizing the functional benefits of peanuts are evident, especially an increase in nicotinate (up to 15-fold) and flavonoids (up to 3.9-fold). Furthermore, the effect of the peanut shell on heat was shown by the different patterns of roasting changes in several compounds, including pyroglutamate, and the changes in amino acids and sugars related to the process of the Maillard reaction were more pronounced

Three-Dimensional Solution Structure of Oryzacystatin-I, a Cysteine Proteinase Inhibitor of the Rice, <i>Oryza sativa</i> L. japonica^†^,^‡

The three-dimensional structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica, has been determined in solution at pH 6.8 and 25 °C by 1H and 15N NMR spectroscopy. The main body (Glu13−Asp97) of oryzacystatin-I is well-defined and consists of an α-helix and a five-stranded antiparallel β-sheet, while the N- and C-terminal regions (Ser2−Val12 and Ala98−Ala102) are less defined. The helix-sheet architechture of oryzacystatin-I is stabilized by a hydrophobic cluster formed between the α-helix and the β-sheet and is considerably similar to that of monellin, a sweet-tasting protein from an African berry, as well as those of the animal cystatins studied, e.g., chicken egg white cystatin and human stefins A and B (also referred to as human cystatins A and B). Detailed structural comparison indicates that oryzacystatin-I is more similar to chicken cystatin, which belongs to the type-2 animal cystatins, than to human stefins A and B, which belong to the type-1 animal cystatins, despite different loop length

Two-Dimensional ¹H–¹³C Nuclear Magnetic Resonance (NMR)-Based Comprehensive Analysis of Roasted Coffee Bean Extract

Coffee was characterized by proton and carbon nuclear magnetic resonance (NMR) spectroscopy. To identify the coffee components, a detailed and approximately 90% signal assignment was carried out using various two-dimensional NMR spectra and a spiking method, in which authentic compounds were added to the roasted coffee bean extract (RCBE) sample. A total of 24 coffee components, including 5 polysaccharide units, 3 stereoisomers of chlorogenic acids, and 2 stereoisomers of quinic acids, were identified with the NMR spectra of RCBE. On the basis of the signal assignment, state analyses were further launched for the metal ion–citrate complexes and caffeine–chlorogenate complexes. On the basis of the signal integration, the coffee components were successfully quantified. This NMR methodology yielded detailed information on RCBE using only a single observation and provides a systemic approach for the analysis of other complex mixtures

Binding of an RNA pol II Ligand to the WW Domain of Pin1 Using Molecular Dynamics Docking Simulations

A novel docking protocol using a long, all atom molecular dynamics (MD) simulation, in an explicit solvent medium, without using any distance constraints is presented. This MD docking protocol is able to dock ligands, based on the C-terminal domain (CTD) of RNA polymerase II, into the tryptophan-tryptophan (WW) domain of Pin1. In this docking process, a significant loop-bending event occurs in order to encircle the ligand into its solvent exposed binding site, which cannot be simulated using current protocols. The simulations were validated structurally and energetically against an X-ray structure to confirm correct sampling of conformational space. Based on these simulations, and justification of the starting structure as a valid intermediate structure, a potential molecular basis for binding was predicted as well as confirming the key residues involved in the formation of the final strong and stable Pin1 WW domain-ligand complex

Distinguishable <i>In Vitro</i> Binding Mode of Monomeric TRBP and Dimeric PACT with siRNA

<div><p>RNA interference (RNAi) is an evolutionally conserved posttranscriptional gene-silencing mechanism whereby small interfering RNA (siRNA) triggers sequence-specific cleavage of its cognate mRNA. Dicer, Argonaute (Ago), and either TAR-RNA binding protein (TRBP) or a protein activator of PKR (PACT) are the primary components of the RNAi pathway, and they comprise the core of a complex termed the RNA-induced silencing complex (RISC)-loading complex (RLC). TRBP and PACT share similar structural features including three dsRNA binding domains (dsRBDs), and a complex containing Dicer and either TRBP or PACT is considered to sense thermodynamic asymmetry of siRNA ends for guide strand selection. Thus, both TRBP and PACT are thought to participate in the RNAi pathway in an indistinguishable manner, but the differences in siRNA binding mode and the functional involvement of TRBP and PACT are poorly understood. Here, we show <i>in vitro</i> binding patterns of human TRBP and PACT to siRNA using electrophoresis mobility shift analysis and gel filtration chromatography. Our results clearly showed that TRBP and PACT have distinct <i>in vitro</i> siRNA binding patterns from each other. The results suggest that monomeric TRBP binds to siRNA at the higher affinity compared to the affinity for own homodimerization. In contrast, the affinity between PACT and siRNA is lower than that of homodimerization or that between TRBP and siRNA. Thus, siRNA may be more readily incorporated into RLC, interacting with TRBP (instead of PACT) <i>in vivo</i>.</p></div

Model of TRBP and PACT binding to siRNA.

<p>(A, B) TRBP-WT (A) or TRBP-ΔdsRBD3 (B) binds one molecule of siRNA as a monomer at low concentrations, and then each protein dimerizes due to the increased protein concentration. However, excessive amount of siRNAs were added, TRBP-WT or TRBP-ΔdsRBD3 dimer was dissociated into monomeric TRBP-WT or TRBP-ΔdsRBD3 dimer containing a single molecule of siRNA. (C) PACT-WT forms homodimers at high concentrations and binds to one or two molecules of siRNA. (D) PACT-ΔdsRBD3 binds one siRNA molecule as a monomer or binds one or two siRNA molecules as a dimer. The monomer and dimer may achieve equilibrium, although the monomeric form is predominant. In C and D, we could not determine whether the siRNA shown in gray is contained in the dimerized PACT proteins or not.</p

Binding of an RNA pol II Ligand to the WW Domain of Pin1 Using Molecular Dynamics Docking Simulations

A novel docking protocol using a long, all atom molecular dynamics (MD) simulation, in an explicit solvent medium, without using any distance constraints is presented. This MD docking protocol is able to dock ligands, based on the C-terminal domain (CTD) of RNA polymerase II, into the tryptophan-tryptophan (WW) domain of Pin1. In this docking process, a significant loop-bending event occurs in order to encircle the ligand into its solvent exposed binding site, which cannot be simulated using current protocols. The simulations were validated structurally and energetically against an X-ray structure to confirm correct sampling of conformational space. Based on these simulations, and justification of the starting structure as a valid intermediate structure, a potential molecular basis for binding was predicted as well as confirming the key residues involved in the formation of the final strong and stable Pin1 WW domain-ligand complex

Roasting Process of Coffee Beans as Studied by Nuclear Magnetic Resonance: Time Course of Changes in Composition

In this paper, we report a ¹H and ¹³C nuclear magnetic resonance (NMR)-based comprehensive analysis of coffee bean extracts of different degrees of roast. The roasting process of coffee bean extracts was chemically characterized using detailed signal assignment information coupled with multivariate data analysis. A total of 30 NMR-visible components of coffee bean extracts were monitored simultaneously as a function of the roasting duration. During roasting, components such as sucrose and chlorogenic acids were degraded and components such as quinic acids, <i>N</i>-methylpyridinium, and water-soluble polysaccharides were formed. Caffeine and <i>myo</i>-inositol were relatively thermally stable. Multivariate data analysis indicated that some components such as sucrose, chlorogenic acids, quinic acids, and polysaccharides could serve as chemical markers during coffee bean roasting. The present composition-based quality analysis provides an excellent holistic method and suggests useful chemical markers to control and characterize the coffee-roasting process