Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions

Reversible control of the conformation of proteins was employed to probe the relationship between flexibility and specificity of the basic helix-loop-helix protein MyoD. A fusion protein (apaMyoD) was designed where the basic DNA binding helix of MyoD was stablized by an amino-terminal extension with a sequence derived from the bee venom peptide apamin. The disulfide-stabilized helix from apamin served as a nucleus for a helix that extended for a further ten residues, thereby holding apaMyoD's DNA recognition helix in a predominantly alpha-helical conformation. The thermal stability of the DNA complexes of apaMyoD was increased by 13 degrees C relative to MyoD-bHLH. Measurements of the fluorescence anisotropy change on DNA binding indicated that apaMyoD bound to E-box-containing DNA sequences with enhanced affinity relative to MyoD-bHLH. Consequently, the DNA binding specificity of apaMyoD was increased 10-fold.     

Fragmentation of N-linked glycans with a matrix-assisted laser desorption/ionization ion trap time-of-flight mass spectrometer

N-Linked glycans were ionized from several matrices with a Shimadzu-Biotech AXIMA-QIT matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer. [M+Na]+ ions were produced from all matrices and were accompanied by varying amounts of in-source fragmentation products. The least fragmentation was produced by 2,5-dihydroxybenzoic acid and the most by alpha-cyano-4-hydroxycinnamic acid and 6-aza-2-thiothymine. Sialic acid loss was extensive but could be prevented by formation of methyl esters. Fragmentation produced typical low-energy-type spectra dominated by ions formed by glycosidic cleavages. MS(n) spectra (n = 3 and 4) were used to probe the pathways leading to the major diagnostic ions. Thus, for example, an ion that was formed by loss of the core GlcNAc residues and the 3-antenna was confirmed as being formed by a B/Y rather than a C/Z mechanism. The proposed structures of several cross-ring cleavage ions were confirmed and it was shown that MS3 spectra could be obtained from as little as 10 fmol of glycan.     

Cluster Chemsitry. 96. Penta- and hexa-nuclear ruthenium cluster complexes derived from reactions of cyclopentadienes with Ru5(μ5-C2PPh2)(μ-PPh2)(CO)13

The reactions between Ru₅( ₅-C₂PPh₂)(gm-PPh₂(CO)₁₃ (1) and cyclopentadienes afforded the hexanuclear clusters Ru₆( ₆-C)( ₃-PPh₂)₂(CO)₁₀(-C₅ R ₅) [R ₅ = H₅ (2), H₄Me (3), Me₅ (4)] which contain an encapsulated carbide and a face-capping ₃-CH group, formed by cleavage of CC and CP bonds of the C₂PPh₂ moiety in1. In the reaction with cyclopentadiene, the unusual ligand C₁₃H₁₂O, formed by combination of C₂, CO and two molecules of C₅H₆ (or one molecule of dicyclopentadien), was characterized in the complex Ru₅( ₄-PPh) ( ₄-C₁₃H₁₂O)(-PPh₂(CO)₁₁(-C₅H₅) (5). In the reaction with pentamethylcyclopentadiene, the vinylidene complex Ru₅( ₃-CCHPh)( ₄-PPh)( ₄-PPh) (-PPh₂)(CO)₉(-C₅Me₅) (6) was also formed.     

Combined first-principles computational and experimental multinuclear solid-state NMR investigation of amino acids

13C, 14N, 15N, 17O, and 35Cl NMR parameters, including chemical shift tensors and quadrupolar tensors for 14N, 17O, and 35Cl, are calculated for the crystalline forms of various amino acids under periodic boundary conditions and complemented by experiment where necessary. The 13C shift tensors and 14N electric field gradient (EFG) tensors are in excellent agreement with experiment. Similarly, static 17O NMR spectra could be precisely simulated using the calculation of the full chemical shift (CS) tensors and their relative orientation with the EFG tensors. This study allows correlations to be found between hydrogen bonding in the crystal structures and the 17O NMR shielding parameters and the 35Cl quadrupolar parameters, respectively. Calculations using the two experimental structures for L-alanine have shown that, while the calculated isotropic chemical shift values of 13C and 15N are relatively insensitive to small differences in the experimental structure, the 17O shift is markedly affected.     

Novel carbohydrate-appended metal complexes for potential use in molecular imaging

Seven discrete sugar-pendant diamines were complexed to the {M(CO)(3)}(+) ((99m)Tc/Re) core: 1,3-diamino-2-propyl beta-D-glucopyranoside (L(1)), 1,3-diamino-2-propyl beta-D-xylopyranoside (L(2)), 1,3-diamino-2-propyl alpha-D-mannopyranoside (L(3)), 1,3-diamino-2-propyl alpha-D-galactopyranoside (L(4)), 1,3-diamino-2-propyl beta-D-galactopyranoside (L(5)), 1,3-diamino-2-propyl beta-(alpha-D-glucopyranosyl-(1,4)-D-glucopyranoside) (L(6)), and bis(aminomethyl)bis[(beta-D-glucopyranosyloxy)methyl]methane (L(7)). The Re complexes [Re(L(1)-L(7))(Br)(CO)(3)] were characterized by (1)H and (13)C 1D/2D NMR spectroscopy which confirmed the pendant nature of the carbohydrate moieties in solution. Additional characterization was provided by IR spectroscopy, elemental analysis, and mass spectrometry. Two analogues, [Re(L(2))(CO)(3)Br] and [Re(L(3))(CO)(3)Br], were characterized in the solid state by X-ray crystallography and represent the first reported structures of Re organometallic carbohydrate compounds. Conductivity measurements in H(2)O established that the complexes exist as [Re(L(1)-L(7))(H(2)O)(CO)(3)]Br in aqueous conditions. Radiolabelling of L(1)-L(7) with [(99m)Tc(H(2)O)(3)(CO)(3)](+) afforded in high yield compounds of identical character to the Re analogues. The radiolabelled compounds were determined to exhibit high in vitro stability towards ligand exchange in the presence of an excess of either cysteine or histidine over a 24 h period.     

Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC.     

Direct observation of the protonation state of an imino sugar glycosidase inhibitor upon binding

Glycosidases are some of the most ubiquitous enzyme in nature. Their biological significance, coupled to their enormous catalytic prowess derived from tight binding of the transition state, is reflected in their importance as therapeutic targets. Many glycosidase inhibitors are known. Imino sugars are often potent inhibitors, yet many facets of their mode of action, such as their degree, if any, of transition-state "mimicry" and their protonation state when bound to the target glycosidase remain unclear. Atomic resolution analysis of the endoglucanase, Cel5A, in complex with a cellobio-derived isofagomine in conjunction with the pH dependence of Ki and kcat/KM reveals that this compound binds as a protonated sugar. Surprisingly, both the enzymatic nucleophile and the acid/base are unprotonated in the complex.     

Highly enantioselective hydrogenation of aromatic-heteroaromatic ketones

Asymmetric hydrogenation of ketone 1 using trans-RuCl(2)[(R)-xylbinap][(R)-daipen] (3) as a catalyst afforded secondary alcohol 2 quantitatively and in 99.4% ee. Further exploration of the effect of the thiazole ring substitution revealed that the catalyst was highly effective for the enantioselective hydrogenation of 5-benzoyl thiazoles, which afforded corresponding alcohols in 92-99% ee. The same protocol was applicable to a variety of aromatic-heteroaromatic ketones to generate secondary alcohols in excellent enantioselectivities. [reaction: see text]