Human plasma afamin, the fourth member of the albumin gene family, is shown to be a specific binding protein for vitamin E. A radio ligand-binding assay followed by Scatchard and Hill analysis are used to show that afamin has a binding affinity for both alpha-tocopherol and gamma-tocopherol, two of the most important forms of vitamin E, in vitro. The binding-dissociation constant was determined to be 18 microM, indicating that afamin plays a role as vitamin E carrier in body fluids such as human plasma and follicular fluid under physiological conditions. Additionally, we demonstrate that afamin has multiple binding sites for both alpha- and gamma-tocopherol. Due to the large binding capacity of afamin for vitamin E, it might take over the role of vitamin E transport in body fluids under conditions where the lipoprotein system is not sufficient for vitamin E transport. To confirm the experimental results, we performed homology modeling and docking calculations on the predicted tertiary structure, which showed coincidence between calculated and in vitro results.

Virtual combinatorial chemistry easily produces billions of compounds, for which conventional virtual screening cannot be performed even with the fastest methods available. An efficient solution for such a scenario is the generation of Fragment Spaces, which encode huge numbers of virtual compounds by their fragments/reagents and rules of how to combine them. Similarity-based searches can be performed in such spaces without ever fully enumerating all virtual products. Here we describe the generation of a huge Fragment Space encoding about 5 * 10(11) compounds based on established in-house synthesis protocols for combinatorial libraries, i.e., we encode practically evaluated combinatorial chemistry protocols in a machine readable form, rendering them accessible to in silico search methods. We show how such searches in this Fragment Space can be integrated as a first step in an overall workflow. It reduces the extremely huge number of virtual products by several orders of magnitude so that the resulting list of molecules becomes more manageable for further more elaborated and time-consuming analysis steps. Results of a case study are presented and discussed, which lead to some general conclusions for an efficient expansion of the chemical space to be screened in pharmaceutical companies.

The development of sequence-specific minor groove binding ligands is a modern and rapidly growing field of research because of their extraordinary importance as transcription-controlling drugs. We performed three molecular dynamics simulations in order to clarify the influence of minor groove binding of two ImHpPyPy-beta-Dp polyamides to the d(CCAGTACTGG)(2) decamer in the B-form. This decamer contains the recognition sequence for the trp repressor (5'-GTACT-3'), and it was investigated recently by X-ray crystallography. On one hand we are able to reproduce X-ray-determined DNA--drug contacts, and on the other hand we provide new contact information which is important for the development of potential ligands. The new insights show how the beta-tail of the polyamide ligands contributes to binding. Our simulations also indicate that complexation freezes the DNA backbone in a specific B(I) or B(II) substate conformation and thus optimizes nonbonded contacts. The existence of this distinct B(I)/B(II) substate pattern also allows the formation of water-mediated contacts. Thus, we suggest the B(I) <==> B(II) substate behavior to be an important part of the indirect readout of DNA.

An algorithm was developed for the computer-aided interpretation of fragment ion spectra from collision-induced dissociation of multiply charged oligodeoxynucleotide ions generated by electrospray ionization. The method compares the experimental spectrum to the m/z values predicted by employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between measured spectrum and predicted m/z values. To substantiate the identity of investigated sequence and reference sequence, or to identify point mutations or insertions/deletions, the reference sequence is systematically varied by incorporating all four possible nucleotides A, T, G, and C at each position in the sequence followed by identification of the correct sequence by the lowest fitness value. Collision energy was shown to have a major impact on the interpretability of the tandem mass spectra by the comparative sequencing algorithm, and the optimal collision energy depended on the length of the fragmented oligodeoxynucleotide. The analytical system was successfully applied to verify DNA sequences as well as to detect and localize point mutations or insertions/deletions in 5-51-mer oligodeoxynucleotides.

We have examined the backbone dynamics of two alternating purine-pyrimidine dodecamers. One sequence consists of "pure" GC bases; the other one contains 5-methylcytosines. The effect of the methyl groups on the backbone substates BI/BII was investigated by means of molecular dynamics. The methylation influences, on one hand, the transition barrier between BI and BII and, on the other hand, the state of equilibrium. The kinetic consequences are an increase of the DeltaG of Gp5mC steps by 1.5 kcal/mol and a decrease of the DeltaG of 5mCpG steps by 0.8 kcal/mol (compared with the nonmethylated DNA). Thus, the additive group differentiates between the two occurring dinucleotide steps and renders the phosphate of the 5-methylcytosine more rigid, as proposed by experimental studies. The thermodynamic consequences are an increase of the DeltaG of Gp5mC steps by 1.1 kcal/mol and a decrease of the DeltaG of 5mCpG steps by 0.8 kcal/mol. The reason for this shift in equilibrium is still not completely clear on a molecular basis. But we can conclude that the indirect readout of DNA is influenced by methylation.