Bonding in HNO-myoglobin as characterized by X-ray absorption and resonance raman spectroscopies

The EXAFS and resonance Raman spectra on the HNO-myoglobin adduct, 1, are consistent with the presence of HNO bound to a heme center. The three-dimensional structure about the heme center of 1 obtained from multiple-scattering (MS) analysis of the EXAFS of the heme protein yielded an Fe-N-O bond angle of 131 degrees and an Fe-N bond length of 1.82 A, which compare well with published values for model complexes containing RNO ligands. Resonance Raman spectra identified the nu(N=O) stretch at 1385 cm-1 (confirmed by 15N labeling), which corresponds well with those reported for small molecule HNO complexes. The wavelength of the nu(Fe-N) at 636 cm-1 of 1 is significantly higher than those of MbIINO and MbIIINO (554 and 595 cm-1, respectively). The XAFS, XANES, and resonance Raman data are all consistent with the structure deduced from the NMR experiments, providing more detail on the bonding between HNO and the metal center.     

Comparison of C-H...pi and hydrophobic interactions in a beta-hairpin peptide: impact on stability and specificity

We have examined the impact of C-H...pi and hydrophobic interactions in the diagonal position of a beta-hairpin peptide through comparison of the interaction of Phe, Trp, or Cha (cyclohexylalanine) with Lys or Nle (norleucine). NMR studies, including NOESY and chemical shift perturbation studies, of the Lys side chain indicates that Lys interacts in a specific geometry with Phe or Trp through the polarized C epsilon. In contrast, Nle does not interact in a specific manner with the diagonal aromatic residue. Thermal denaturation provides additional support that Lys and Nle interact in fundamentally different manners. Folding of the peptide with a diagonal Trp...Lys interaction was found to be enthalpically driven, whereas the peptide with a diagonal Trp...Nle interaction displayed cold denaturation, as did the control peptide with a diagonal Cha...Nle interaction, indicating different driving forces for interaction of Lys and Nle with Trp. These findings have significant implications for specificity in protein folding and de novo protein design.     

Synthesis and structural characterization of bi- and trimetallic octacyanometalate(IV) complexes: [Delta,Lambda-MII(en)3][cis-MII(en)2(OH2)][MIV(CN)8].2H2O and [cis-MII(en)2(OH2)]2[(mu-NC)2MIV(CN)6].4H2O (MII = Mn, Co, Ni; MIV = Mo, W)

Treatment of [M(II)(en)(3)][OTs](2) or methanolic ethylenediamine solutions containing transition metal p-toluenesulfonates (M(II) = Mn, Co) with aqueous K(4)M(IV)(CN)(8).2H(2)O or Cs(3)M(V)(CN)(8) (M(IV) = Mo, W; M(V) = Mo) affords crystalline clusters of [M(II)(en)(3)][cis-M(II)(en)(2)(OH(2))(mu-NC)M(IV)(CN)(7)].2H(2)O (M(IV) = Mo; M(II) = Mn, 1; Ni, 5; M(IV) = W; M(II) = Mn, 2; Ni, 6) and [cis-M(II)(en)(2)(OH(2))](2)[(mu-NC)(2)M(IV)(CN)(6)].4H(2)O (M(IV) = Mo; M(II) = Co, 3; Ni, 7; M(IV) = W; M(II) = Co, 4) stoichiometry. Each cluster contains cis-M(II)(en)(2)(OH(2))(mu-NC)(2+) units that likely result from dissociative loss of en from [M(II)(en)(3)](2+), affording cis-M(II)(en)(2)(OH(2))(2)(2+) intermediates that are trapped by M(IV)(CN)(8)(4-).     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

Moving beyond molecules: patterning solid-state features via dip-pen nanolithography with sol-based inks

Herein, we described a new dip-pen nanolithography (DPN)-based method for the direct patterning of organic/inorganic composite nanostructures on silicon and oxidized silicon substrates. The approach works by the hydrolysis of metal precursors in the meniscus between an AFM tip and a surface according to the reaction 2MCln + nH2O --> M2On + 2nHCl; M = Al, Si, and Sn. The inks are hybrid composites of inorganic salts with amphiphilic block copolymer surfactants. Three proof-of-concept systems involving Al2O3, SiO2, and SnO2 nanostructures on silicon and silicon oxide surfaces have been studied. Arrays of dots and lines can be written easily with control over feature size and shape on the sub-200 nm level. The structures have been characterized by atomic force microscopy, scanning electron microscopy, transmission electron microscopy, and energy-dispersive X-ray analysis. This work is important because it opens up the opportunity for using DPN to deposit solid-state materials rather than simple organic molecules onto surfaces with the resolution of an AFM without the need for a driving force other than chemisorption (e.g., applied fields).     

Automated data interpretation based on the concept of "negative signature mass" for mass-mapping disulfide structures of cystinyl proteins

An efficient method for data processing and interpretation is needed to support and extend disulfide mass-mapping methodology based on partial reduction and cyanylation-induced cleavage to proteins containing more than four cystines. Here, the concept of "negative signature mass" is introduced as the novel feature of an algorithm designed to identify the disulfide structure of a cystinyl protein given an input of mass spectral data and an amino acid sequence. The "negative signature mass" process is different from the conventional approach in that it does not directly rule-in disulfide linkages, but rather eliminates linkages from a list of all possible theoretical linkages, with the goal of ruling out enough linkages so that only one disulfide structure can be constructed. The operating principles and the effectiveness of the algorithm are described in the context of analyzing ribonuclease A, a 124-residue protein containing eight cysteines in the form of four cystines (disulfides).     

Binuclear ruthenium macrocycles formed via the weak-link approach

The "weak-link approach" for the synthesis of metallomacrocycles has been used to synthesize a series of novel Ru(II) macrocycles in high yield. RuCl2(PPh3)3 has been reacted with two different phosphino-alkyl-ether hemilabile ligands, 1,4-(PPh2(CH2)2O)2C6H4 and 1,4-(PPh2(CH2)2OCH2)2C6H4. The hemilabile bidentate ligand coordinates to Ru(II) centers through both the P and O atoms to form bimetallic "condensed intermediates". The weak Ru-O bonds have been selectively cleaved with CO, 1,2-diaminopropane, and pyridine to yield large open macrocycles. This is the first example of the weak-link approach employed to synthesize macrocycles with Ru, and metal centers in general that have more than four coordination sites.     

On the structure and desorption dynamics of DNA bases adsorbed on gold: a temperature-programmed study

The structure and desorption dynamics of mono- and multilayer samples of adenine, cytosine, guanine, and thymine on polycrystalline gold thin films are studied using temperature-programmed desorption-infrared reflection absorption spectroscopy (TPD-IRAS) and temperature-programmed desorption-mass spectroscopy (TPD-MS). It is shown that the pyrimidines, adenine and guanine, adsorb to gold in a complex manner and that both adhesive (adenine) and cohesive (guanine) interactions contribute the apparent binding energies to the substrate surface. Adenine displays at least two adsorption sites, including a high-energy site (210 degrees C, approximately 136 kJ/mol), wherein the molecule coordinates to the gold substrate via the NH2 group in an sp3-like, strongly perturbed, nonplanar configuration. The purines, cytosine and thymine, display a less complicated adsorption/desorption behavior. The desorption energy for cytosine (160 degrees C, approximately 122 kJ/mol) is similar to those obtained for adenine and guanine, but desorption occurs from a single site of dispersed, nonaggregated cytosine. Thymine desorbs also from a single site but at a significantly lower energy (100 degrees C, approximately 104 kJ/mol). Infrared data reveal that the monolayer architectures discussed herein are structurally very different from those observed for the bases in the bulk crystalline state. It is also evident that both pyrimidines and purines adsorb on gold with the plane of the molecule in a nonparallel orientation with respect to the substrate surface. The results of this work are discussed in the context of improving the understanding of the design of capturing oligonucleotides or DNA strands for bioanalytical applications, in particular, for gold nanoparticle-based assays.     

Real-time multicolor DNA detection with chemoresponsive diffraction gratings and nanoparticle probes

We report a real-time DNA detection method that utilizes single-strand DNA-modified nanoparticle probes and micropatterned chemoresponsive diffraction gratings interrogated simultaneously at multiple laser wavelengths. The surface-bound nanoparticle probe based assay with the chemoresponsive diffraction grating signal transduction scheme results in an experimentally simple DNA detection protocol, displaying attributes of both detection methodologies: the high sensitivity and selectivity afforded by nanoparticle probes and the experimental simplicity, wavelength-dependent resonant enhancement features, and miniaturization potential provided by the diffraction-based sensing technology.     

Tellurium-mediated cycloaromatization of acyclic enediynes under mild conditions

The cycloaromatization of acyclic enediynes typically requires very high temperatures (>160 degrees C) and dilute conditions to proceed in a synthetically useful yield. These conditions hinder reaction throughput, inhibiting the use of this reaction for the large-scale production of materials. The reaction of sodium telluride with acyclic arenediynes yields the corresponding tellurepine, which under gentle heating extrudes Te degrees to yield the cycloaromatization product. We have developed conditions that form sodium telluride from inexpensive tellurium metal in situ, and that also perform the desilylation of silylated arenediynes in the same process. Under our conditions, we are able to perform desilylation and cycloaromatization at temperatures as low as 40 degrees C and on a scale as large as 5 g in standard laboratory glassware.