A phosphoramidite of the perylene bisimide dye was synthesized as a DNA building block that allows incorporation of this chromophore as an artificial nucleoside surrogate either at the 5'-terminus or at internal positions of duplex DNA. The internally incorporated perylene bisimide chromophore shows strong interactions with the DNA base stack; the 5'-terminally attached perylene bisimide is able to induce dimerization of two whole DNA duplexes.

A new DNA assay has been designed, prepared and applied for the chemical investigation of reductive electron transfer through the DNA. It consists of 5-(10-methyl-phenothiazin-3-yl)-2'-deoxyuridine (Ptz-dU, 1) as the photoexcitable electron injector and 5-bromo-2'-deoxyuridine (Br-dU) as the electron trap. The Ptz-dU-modified oligonucleotides were synthesised by means of a Suzuki-Miyaura cross-coupling protocol and subsequent automated phosphoramidite chemistry. Br-dU represents a kinetic electron trap, since it undergoes a chemical modification after its one-electron reduction that can be analysed by piperidine-induced strand cleavage. The quantification of the strand cleavage yields from irradiation experiments reveals important information about the electron-transfer efficiency. The performed DNA studies focused on the base sequence dependence of the electron-transfer efficiency with respect to the proposal that C*- and T*- act as intermediate electron carriers during electron hopping. From our observations it became evident that excess-electron transfer is highly sequence dependent and occurs more efficiently over T-A base pairs than over C-G base pairs.

Hence, we propose to instead focus on the conversion cone, a projection of the flux cone, which describes the interaction of the metabolism with its external chemical environment. We present a direct method for calculating the elementary vectors of this cone and, by studying the metabolism of Saccharomyces cerevisiae, we demonstrate that such an analysis is computationally feasible even for genome scale networks.

Mathematically, the calculation of elementary fluxes amounts to characterizing the space of solutions to a mixed system of linear equalities, given by the stoichiometry matrix, and linear inequalities, arising from the irreversibility of some or all of the reactions in the network. Previous approaches to this problem have iteratively solved for the equalities while satisfying the inequalities throughout the process. In an extension of previous work, here we consider the complementary approach and derive an algorithm which satisfies the inequalities one by one while staying in the space of solution of the equality constraints. Benchmarks on different subnetworks of the central carbon metabolism of Escherichia coli show that this new approach yields a significant reduction in the execution time of the calculation. This reduction arises since the odds that an intermediate elementary flux already fulfills an additional inequality are larger than when having to satisfy an additional equality constraint.

The effect of intermediates on the rate of protein folding is explored by applying Kramers' theory of diffusive barrier crossing in the high friction limit. Intermediates are represented as local minima in the transition barrier. We observe that very large or very small additional barriers created by the intermediates slow down the folding process. The rate of folding markedly increases, however, when the additional barriers become >1 k B T but leave the overall barrier height unchanged. This rate-enhancing effect is caused by a favorable entropic contribution to the free energy of activation, and it increases with the number of intermediates up to a limiting value. From these calculations, we conclude that optimized transition barriers should contain partially folded high energy intermediates.Various models have been proposed to describe the mechanism of protein folding. The experimental observation of transiently populated, partially folded intermediates in many proteins gave rise to the framework model, which assumes that the native structure is formed in a hierarchical way on a linear pathway involving several consecutive transition states (1, 2). In this model, partially folded intermediates are essential for protein folding by directing the chain to the native state. In theoretical approaches, the folding process is conceived as a movement of molecules on a rough, funnel-like energy landscape starting from the ensemble of unfolded conformations and leading to the native state (3-5). In these models, transiently populated intermediates often represent misfolded structures trapped in local energy minima.Based on these opposing views, the study of the role of protein folding intermediates has been of major interest in theoretical and experimental work. Recent experimental results provided evidence for the presence of metastable, high energy states located in the transition barrier between the native state and the ensemble of unfolded molecules. Native state hydrogen exchange studies revealed partially unfolded states in cytochrome c (6) and RNase H (7), which are higher in energy than the native protein. Although these intermediates were identified as fluctuations from the native structure under equilibrium conditions, it was postulated that they might represent intermediates on linear folding pathways (6). Local energy minima in the transition barrier also were observed in unfolding reactions of fast-folding proteins that reach the native state without transient population of partially folded intermediates. For the dimeric arc repressor (8) and staphylococcal nuclease (9), a nonlinearity in the denaturant dependence of the free energy of activation for the unfolding reaction (⌬G u 0 ‡ ) was interpreted as evidence for two distinct transition states on a sequential pathway. A similar observation was made for the formation of a helical intermediate in lysozyme folding, which proceeds through a reactive high energy intermediate (10). For chymotrypsin inhibitor 2, a pronounced curvatures in the denaturan...