Analysis of high molecular mass proteins larger than 150 kDa using cyanogen bromide cleavage and conventional 2-DE

Proteomic approaches including high-resolution 2-DE are providing the tools needed to discover disease-associated biomarkers in complex biological samples. Although 2-DE is an extremely powerful approach to analyze the proteome, the separation of proteins with extreme molecular masses still remains an issue requiring improvement. Because high molecular mass (HMM) proteins larger than 150 kDa have already been observed to be differentially expressed in several pathologies such as cancer, we developed an original strategy to analyze this part of the proteome that is not easily separated by 2-DE in polyacrylamide gels. This strategy is based on the 2-DE separation of cyanogen bromide (CNBr) fragments of purified HMM protein fractions, and combines techniques including SEC fractionation, TCA precipitation, CNBr cleavage, 2-DE and MS analysis. The method was first tested on a model protein, the BSA. Preliminary results obtained using colonic tissues led to the identification of six HMM proteins with M(r) comprised between 163 and 533 kDa in their reduced state. These results demonstrated that our CNBr/2-DE approach should provide a powerful tool for identification of new biomarkers larger than 150 kDa.     

Experimental and computational study of the conrotatory ring opening of various 3-chloro-2-azetines

A combined experimental and theoretical study is presented on 2-azetines, a class of azaheterocyclic compounds, which are difficult to access but have shown a unique reactivity as strained cyclic enamines. New highly substituted 2-azetines bearing aryl substituents at the 2- and 4-position were synthesized from 3,3-dichloroazetidines. Whereas 2-aryl-3,3-dichloroazetidines gave stable 2-aryl-3-chloro-2-azetines upon treatment with sodium hydride in DMSO, 2,4-diaryl-3,3-dichloroazetidines showed a remarkably different reactivity in that they afforded benzimidoyl-substituted alkynes under similar mild treatment with base. The formation of the alkynes involves electrocyclic ring opening of intermediate 2,4-diaryl-3-chloro-2-azetines and elimination of hydrogen chloride. Ab initio theoretical calculations confirmed the experimental findings and demonstrated that the 4-aryl substituent is responsible for this remarkably enhanced reactivity of 2-azetines toward electrocyclic conrotatory ring opening by a significant decrease in reaction barrier of about 30 kJ/mol. This activation effect by an aryl group in the allylic position toward electrocyclic ring opening of unsaturated four-membered rings is of general importance since a similar increased reactivity of 4-aryloxetes, 4-arylthiete-1,1-dioxides, and 3-arylcyclobutenes has been reported in literature as well.     

Aza-beta3-cyclotetrapeptides

The cyclization of aza-beta(3)-tetrapeptides gives access to new CTP (cyclotetrapeptide) analogues. These stereocontrolled templates are assembled without any asymmetric synthesis. X-ray crystallographic structure and NMR analysis show that the macrocyclic scaffold is characterized by a fully cooperative intramolecular H-bond network, in sharp contrast with the nanotubular assemblies observed for beta(3)-cyclotetrapeptides. This folding property reduces considerably the polarity of aza-beta(3)-tetrapeptides and should be useful in addressing intracellular targets.     

Optimized and far-red-emitting variants of fluorescent protein eqFP611

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.     

Can electrospray ionization help the characterization of iron gall inks? Investigation on the interactions of gallic acid with iron ions in aqueous solutions

In the present work, an investigation has been conducted by electrospray ionization (ESI) experiments to characterize the structures of iron gall ink complexes in solution. Simple mono and polyphenolic acid molecules added to iron sulfate salts were chosen to model the recipes of ink composition. Theoretical calculations have been used (1) to determine the stability of the ionic complexes generated in the gas phase, (2) to explain which structures are more likely generated in the electrospray ion source, and (3) to determine which mechanisms are likely involved in their formation. Fragmentation pathways of the derived structures have also been investigated and rationalized to facilitate the interpretation of the data obtained under collisionally induced dissociation (CID) conditions.The present study confirms the assumption that ESI experiments with ions that are preformed in solution must be considered carefully. As a matter of fact, the study of ion formation mechanisms in the ion source is necessary to establish relationships between the ion structures in the condensed phase and the gas phase.     

One-electron-transfer reactions of polychlorinated ethylenes: concerted and stepwise cleavages

Reaction barriers were calculated by using ab initio electronic structure methods for the reductive dechlorination of the polychlorinated ethylenes: C2Cl4, C2HCl3, trans-1,2-C2H2Cl2, cis-1,2-C2H2Cl2, 1,1-C2H2Cl2 and C2H3Cl. Concerted and stepwise cleavages of R-Cl bonds were considered. Stepwise cleavages yielded lower activation barriers than concerted cleavages for the reduction of C2Cl4, C2HCl3, and trans-1,2-C2H2Cl2 for strong reducing agents. However, for typical ranges of reducing strength concerted cleavages were found to be favored. Both gas-phase and aqueous-phase calculations predicted C2Cl4 to have the lowest reaction barrier. Additionally, the reduction of C2HCl3 was predicted to show selectivity toward formation of cis-1,2-C2HCl2* over the formation of trans-1,2-C2HCl2*, and 1,1-C2HCl2* radicals.     

Convergence of classes of high-order semi-Lagrangian schemes for the Vlasov-Poisson system

In this paper we present some classes of high-order semi-Lagran- gian schemes for solving the periodic one-dimensional Vlasov-Poisson system in phase-space on uniform grids. We prove that the distribution function and the electric field converge in the norm with a rate of

where is the degree of the polynomial reconstruction, and and are respectively the time and the phase-space discretization parameters.

     

Modeling procyanidin self-association processes and understanding their micellar organization: a study by diffusion NMR and molecular mechanics

The colloidal behavior of eight synthetic procyanidins (three monomers, four dimers, and a trimer) has been investigated in water or in a winelike medium using DOSY NMR spectroscopy and molecular dynamics simulations. Different behavior was observed for monomers and oligomers. Monomers self-associate with a high affinity constant (37-53 M(-1)) to form micelles at low cmc (critical micelle concentration) values (1-5 g.L(-1)). These micelles undergo a time-dependent coalescence process to form hazes and precipitates. As for dimers and the trimer, self-association also occurs but with a lower affinity (approximately 6 M(-1)) and at higher cmc values (10-20 g.L(-1)) to form small micelles (<5 nm) that remain stable throughout the experiment. The presence of 10% ethanol does not significantly affect the self-association constant for monomers and oligomers but increases their cmc values by approximately 50% and decreases the micelle size by a factor 2. However, the presence of 20 mM NaCl appears to negate the effect of ethanol. This study helps to clarify the role of procyanidin monomers versus oligomers in wine turbidity and demonstrates that procyanidin oligomers are fully available to interact with saliva proteins.