Kenji Kanaori

Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: the structural basis of this difference

Through binding and fluorescence studies of oligonucleotides covalently attached to a pyrene group via one carbon linker at the sugar residue, we previously found that pyrene-modified RNA oligonucleotides do not emit well in the single-stranded form, yet the attached pyrene emits with a significantly high quantum yield upon binding to a complementary RNA strand. In sharp contrast, similarly modified pyrene–DNA probes exhibit very weak fluorescence both in the double-stranded and single-stranded forms. The pyrene-modified RNA oligonucleotides therefore provide a useful tool for monitoring RNA hybridization. The purpose of this paper is to present the structural basis for the different fluorescence properties of pyrene-modified RNA/RNA and pyrene-modified DNA/DNA duplexes. The results of absorption, fluorescence anisotropy and circular dichroism studies all consistently indicated that the pyrene attached to the RNA duplex is located outside of the duplex, whereas the pyrene incorporated into the DNA duplex intercalates into the double helix. 1H NMR measurements unambiguously confirmed that the pyrene attached to the DNA duplex indeed intercalates between the base pairs of the duplex. Molecular dynamics simulations support these differences in the local structural elements around the pyrene between the pyrene–RNA/RNA and the pyrene–DNA/DNA duplexes.

Creation of a type 1 blue copper site within a de novo coiled-coil protein scaffold.

Type 1 blue copper proteins uniquely coordinate Cu(2+) in a trigonal planar geometry, formed by three strong equatorial ligands, His, His, and Cys, in the protein. We designed a stable Cu(2+) coordination scaffold composed of a four-stranded α-helical coiled-coil structure. Two His residues and one Cys residue were situated to form the trigonal planar geometry and to coordinate the Cu(2+) in the hydrophobic core of the scaffold. The protein bound Cu(2+), displayed a blue color, and exhibited UV-vis spectra with a maximum of 602-616 nm, arising from the thiolate-Cu(2+) ligand to metal charge transfer, depending on the exogenous axial ligand, Cl(-) or HPO(4)(2-). The protein-Cu(2+) complex also showed unresolved small A(∥) values in the electron paramagnetic resonance (EPR) spectral analysis and a 328 mV (vs normal hydrogen electrode, NHE) redox potential with a fast electron reaction rate. The X-ray absorption spectrum revealed that the Cu(2+) coordination environment was identical to that found in natural type 1 blue copper proteins. The extended X-ray absorption fine structure (EXAFS) analysis of the protein showed two typical Cu-N(His) at around 1.9-2.0 Å, Cu-S(Cys) at 2.3 Å, and a long Cu-Cl at a 2.66 Å, which are also characteristic of the natural type 1 blue copper proteins.

How does hemoglobin generate such diverse functionality of physiological relevance

The absolute values of the O2-affinities (P50, Klow, and Khigh) of hemoglobin (Hb) are regulated neither by changes in the static T-/R-quaternary and associated tertiary structures nor the ligation states. They are pre-determined and regulated by the extrinsic environmental factors such as pH, buffers, and heterotropic effectors. The effect and role of O2 on Hb are reversibly to drive the structural allosteric equilibrium between the T(deoxy)- and R(oxy)-Hb toward R(oxy)-Hb (the structural allostery). R(oxy)-Hb has a higher O2-affinity (Khigh) relative to that (Klow) of the T(deoxy)-Hb (Khigh>Klow) under any fixed environmental conditions. The apparent O2-affinity of Hb is high, as the globin matrix interferes with the dissociation process of O2, forcing the dissociated O2 geminately to re-bind to the heme Fe. This artificially increases [oxy-Hb] and concomitantly decreases [deoxy-Hb], leading to the apparent increases of the O2-affinity of Hb. The effector-linked high-frequency thermal fluctuations of the globin matrix act as a gating mechanism to modulate such physical, energetic, and kinetic barriers to enhance the dissociation process of O2, resulted in increases in [deoxy-Hb] and concomitant decrease in [oxy-Hb], leading to apparent reductions of the O2-affinity of Hb (the entropic allostery). The heme in Hb is simply a low-affinity O2-trap, the coordination structure of which is not altered by static T-/R-quaternary and associated tertiary structural changes of Hb. Thus, heterotrophic effectors are the signal molecule, which acts as a functional link between these two allosteries and generates the diverse functionality of Hb of physiological relevance. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Creation of a binuclear purple copper site within a de novo coiled-coil protein.

Although various kinds of metal binding proteins have been constructed by de novo design, the creation of a binuclear metal binding site remains especially challenging. The purple copper site in subunit II of COX, referred to as the Cu(A) site, has two copper ions bridged by two Cys residues. We constructed the Cu(A) site consisting of two Cys and two His residues in a de novo designed four-helical coiled-coil protein. The protein bound two copper ions and exhibited a purple color, with relatively intense absorption bands at 488 and 530 nm in the UV-vis spectrum. The EPR spectrum displayed unresolved hyperfine splittings in the g(∥) region, which was similar to the native or engineered Cu(A) site with an A(∼480)/A(∼530) > 1. The extended X-ray absorption structure analyses of the protein revealed the presence of the Cu(2)S(2) core structure, with two typical N(His)-Cu bonds per core at 1.90 Å, two S (Cys)-Cu bonds at 2.21 Å, and the Cu-Cu bond at 2.51 Å, which are also characteristic structures of a purple copper site.

Interaction of gymnemic acid with cyclodextrins analyzed by isothermal titration calorimetry, NMR and dynamic light scattering

The physiological phenomenon that the antisweet taste effect of gymnemic acid (GA) is diminished by application of γ‐cyclodextrin (γ‐CD) to the mouth was evaluated at the molecular level using isothermal titration calorimetry, NMR and dynamic light scattering. These analyses showed that GA specifically binds to γ‐CD. Thermodynamic analysis using isothermal titration calorimetry revealed that the association constant of GA and γ‐CD is 105−106 m−1 with favorable enthalpy and entropy changes. The heat capacity change was negative and large, despite the change in accessible surface area upon binding being small. These thermodynamics indicate that the binding is dominated by hydrophobic interactions, which is in agreement with inclusion complex formation of γ‐CD. In addition, NMR measurements showed that in solution the spectra of GA are broad and sharpened by the addition of γ‐CD, indicating that unbound GA is in a water‐soluble aggregate that is dispersed when it forms a complex with γ‐CD. Dynamic light scattering showed that the average diameter of unbound GA is > 30 nm and that of GA and γ‐CD complex is 2.2 nm, similar to unbound γ‐CD, supporting the aggregate property of GA and the inclusion complexation of GA by γ‐CD.

A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase

The radical scavenger 2,2,6,6‐tetramethylpiperidinyl‐1‐oxy (TEMPO•) and the spin trap 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein‐based radical sites of the H2O2‐tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H2O2‐sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO• and DMPO in an H2O2‐dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H2O2. TEMPO• bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine‐to‐serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H2O2, both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H2O2‐mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013–3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.

UV luminescent organic-capped ZnO quantum dots synthesized by alkoxide hydrolysis with dilute water

A novel synthesis route to organic-capped and colloidal ZnO quantum dots (QDs) has been developed. Specifically, zinc-di-t-butoxide and zinc-di-n-butoxide are hydrolyzed by very dilute water (400-600 mass ppm) in hydrophilic benzylamine and polymerized to ZnO by dehydration and/or a butanol elimination reaction. Growth of the ZnO QDs and exchange of the surface capping ligand from the hydroxyl groups and/or benzylamine to the oleylamine occur by heating the colloidal solution after addition of the oleylamine at 100-180°C. The final ZnO QDs with diameters in the range of 3-7 nm are highly dispersible in various organic solvents. The ZnO QDs exhibit the quantum size effect upon UV emission; it was controlled between 3.39 and 3.54 eV in the present study. The defect-related Vis emission decreased and the UV emission becomes dominant when zinc-di-n-butoxide with a 99.99% zinc purity is used as the starting material. The intensity of the photoluminescence UV emission is 1.5 times higher than that of the Vis emission.

Engineering of the hydrophobic core of an α-helical coiled coil

The amino acid sequence that forms the alpha-helical coiled coil structure has a representative heptad repeat denoted by defgabc, according to their positions. Although the a and d positions are usually occupied by hydrophobic residues, hydrophilic residues at these positions sometimes play important roles in natural proteins. We have manipulated a few amino acids at the a and d positions of a de novo designed trimeric coiled coil to confer new functions to the peptides. The IZ peptide, which has four heptad repeats and forms a parallel triple-stranded coiled coil, has Ile at all of the a and d positions. We show three examples: (1) the substitution of one Ile at either the a or d position with Glu caused the peptide to become pH sensitive; (2) the metal ion induced alpha-helical bundles were formed by substitutions with two His residues at the d and a positions for a medium metal ion, and with one Cys residue at the a position for a soft metal ion; and (3) the AAB-type heterotrimeric alpha-helical bundle formation was accomplished by a combination of Ala and Trp residues at the a positions of different peptide chains. Furthermore, we applied these procedures to prepare an ABC-type heterotrimeric alpha-helical bundle and a metal ion-induced heterotrimeric alpha-helical bundle.

1H-NMR and Circular Dichroism Spectroscopic Studies on Changes in Secondary Structures of the Sodium Channel Inactivation Gate Peptides as Caused by the Pentapeptide KIFMK

The pentapeptide KIFMK, which contains three clustered hydrophobic amino acid residues of isoleucine, phenylalanine, and methionine (IFM) in the sodium channel inactivation gate on the cytoplasmic linker between domains III and IV (III-IV linker), is known to restore fast inactivation to the mutant sodium channels having a defective inactivation gate or to accelerate the inactivation of the wild-type sodium channels. To investigate the docking site of KIFMK and to clarify the mechanisms for restoring the fast inactivation, we have studied the interactions between KIFMK and the fragment peptide in the III-IV linker GGQDIFMTEEQK (MP-1A; G1484-K1495 in rat brain IIA) by one- and two-dimensional (1)H-NMR and circular dichroism (CD) spectroscopies. KIFMK was found to increase the helical content of MP-1A in 80% trifluoroethanol (TFE) solution by approximately 11%. A pentapeptide, KIFMT, which can restore inactivation but less effectively than KIFMK, also increased the helical content of MP-1A, but to a lesser extent ( approximately 6%) than did KIFMK. In contrast, KDIFMTK, which is ineffective in restoring inactivation, decreased the helical content ( approximately -4%). Furthermore, we studied the interactions between KIFMK and modified peptides from MP-1A, that is, MP-1NA (D1487N), MP-1QEA (E1492Q), or MP-1EQA (E1493Q). The KIFMK was found to increase the helical content of MP-1EQA to an extent nearly identical to that of MP-1A, whereas it was found to decrease those of MP-1NA and MP-1QEA. These findings mean that KIFMK, by allowing each of the Lys residues to interact with D1487 and E1492, respectively, stabilized the helical structure of the III-IV linker around the IFM residues. This helix-stabilizing effect of KIFMK on the III-IV linker may restore and/or accelerate fast inactivation to the sodium channels having a defective inactivation gate or to wild-type sodium channels.

NMR Spectra of Cyclic Formals Formed during the Early Stage of the Copolymerization of Trioxane and Ethylene Oxide

During the early stage of the copolymerization of trioxane and ethylene oxide, we found the formation of three novel cyclic compounds: 1,3,5,7-tetraoxacyclononane (TOCN), 1,3,5,7,10-pentaoxacyclododecane (POCD), and 1,3,5,7,10,13-hexaoxacyclopentadecane (HOCP). These novel cyclic compounds were new direct reaction products of 1 mole of trioxane and 1 mole of ethylene oxide, 1 mole of trioxane and 2 moles of ethylene oxide, and 1 mole of trioxane and 3 moles of ethylene oxide, respectively. We compared the 1H-NMR and 13C-NMR spectra of each cyclic compound and precisely assigned the signals of each spectrum using NOESY (nuclear Overhauser enhancement spectroscopy) and HETCOR (heteronuclear correlated spectroscopy). We also compared the 1H-NMR spectra of POCD and HOCP with the corresponding cyclic formals with one oxymethylene unit, diethylene glycol formal (DEGF) and triethylene glycol formal (TEGF). Interestingly, we found that DEGF and TEGF, which have only one oxymethylene unit, showed no proton splitting of the oxyethylene units, while POCD and HOCP, which have three consecutive oxymethylene units, have split signals for the oxyethylene units.