The capacity of the flavan-3-ols [(-)-epicatechin (EC) and (+)-catechin (CT)] and a B dimeric procyanidin (DP-B) to modulate phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB activation in Jurkat T cells was investigated. The classic PMA-triggered increase in cell oxidants was prevented when cells were preincubated for 24 h with EC, CT, or DP-B (1.7-17.2 microM). PMA induced the phosphorylation of IKKbeta and the subsequent degradation of IkappaBalpha. These events were inhibited in cells pretreated with the flavonoids. PMA induced a 4.6-fold increase in NF-kappaB nuclear binding activity in control cells. Pretreatment with EC, CT, or DP-B decreased PMA-induced NF-kappaB binding activity and the transactivation of the NF-kappaB-driven gene IL-2. EC, CT, and DP-B inhibited, in vitro, NF-kappaB binding to its DNA consensus sequence, but they had no effect on the binding activity of CREB or OCT-1. Thus, EC, CT, or DP-B can influence the immune response by modulating NF-kappaB activation. This modulation can occur at early (regulation of oxidant levels, IKK activation) as well as late (binding of NF-kappaB to DNA) stages of the NF-kappaB activation cascade. A model is presented for possible interactions between DP-B and NF-kappaB proteins, which could lead to the inhibition of NF-kappaB binding to kappaB sites.

We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.Here, we report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD; Fig. 1, compound 1) an easily prepared cysteine-specific hydrophilic reagent useful for reversibly conjugating ethylenediaminetriacetic acid to any free thiol group in a macromolecule. This reagent is a versatile tool that promotes intramolecular and localized protein cleavage. Staphylococcal nuclease was genetically engineered to introduce a single cysteine at position 28, and this variant, K28C, was used to characterize EPD-Fe as a protein cleavage reagent. Experimentation with a protein of known three-dimensional structure (20, 21) allowed the accessibility and proximity of cleavage sites to be assessed. Sequence analysis of K28C-EDTA-Fe fragmentation products identified several cleavage sites located close in tertiary structure to the reagent attachment site. In the presence of sodium dodecyl sulfate (SDS) or guanidinium chloride, cleavage at sites remote in linear sequence was not observed and the reaction was limited to residues proximal in sequence to the Cys-28 attachment site.There is an increasing need for new probes to study the topology of protein nonnative states, such as the molten globule and other folding intermediates, that can be populated at equilibrium (1, 2). Modem NMR techniques are revolutionizing the analysis of small proteins in solution, but they can be used to characterize partially folded molecules only under favorable conditions (3). Antibodies, proteases, and chemical probes have been used in the study of protein folding (4-8), but they are not well suited for mapping partially folded structure.A new class of chemical probes was devised largely for footprinting studies of DNA (9, 10). These reagents generate reactive oxygen species that label surrounding structural elements by oxidative degradation. Several metal chelates, bound covalently (11-13) or by affinity (14-16) to a protein, are...

After evaluating two current methods, we demonstrate how the use of sequence-weighting techniques to reduce sequence redundancy and low-count corrections to account for small number of observations in limited size sequence families, can significantly improve the predictability of MI. The evaluation is made on large sets of both in silico-generated alignments as well as on biological sequence data. The methods included in the analysis are the APC (average product correction) and RCW (row-column weighting) methods. The best performing method was APC including sequence-weighting and low-count corrections. The use of sequence-permutations to calculate a MI rescaling is shown to significantly improve the prediction accuracy and allows for direct comparison of information values across protein families. Finally, we demonstrate how a lower bound of 400 sequences <62% identical is needed in an MSA in order to achieve meaningful predictive performances. With our contribution, we achieve a noteworthy improvement on the current procedures to determine coevolution and residue contacts, and we believe that this will have potential impacts on the understanding of protein structure, function and folding.