Ultrafast fluorescence decays of FMN binding proteins (FBP) from Desulfovibrio vulgaris (Miyazaki F) were analyzed with an electron transfer (ET) theory by Kakitani and Mataga (KM theory). Time-dependent distances among isoalloxazine (Iso) and Trp-32, Tyr-35, and Trp-106 in wild-type FBP (WT), among Iso and Tyr-32, Tyr-35, and Trp-106 in W32Y (Trp-32 was replaced by Tyr-32), and among Iso and Tyr-35 and Trp-106 in W32A (Trp-32 was replaced by Ala-32) were determined by molecular dynamic simulation (MD). Electrostatic energies between Iso anion and all other ionic groups, between Trp-32 cation and all other ionic groups, and between Tyr-32 cation and all other ionic groups were calculated in WT, W32Y, and W32A, from the MD coordinates. ET parameters contained in KM theory, such as frequency (nu 0), a coefficient of the ET process (beta), a critical distance of the ET process ( R 0), standard free energy related to the electron affinity of the excited Iso ( G Iso (0)), and the static dielectric constant in FBP species (epsilon 0), were determined with and without inclusion of the electrostatic energy, so as to fit the calculated fluorescence decays with the observed decays of all FBP species, by a nonlinear least-squares method according to the Marquardt algorithm. In the analyses the parameters, nu 0, beta, and R 0 were determined separately between Trp residues and Tyr residues among all FBP species. Calculated fluorescence intensities with the inclusion of the electrostatic energy fit quite well with the observed ones of all WT, W32Y, and W32A.

The pathogenic West Nile virus (WNV) and Dengue virus (DV) are growing global threats for which there are no specific treatments. Both viruses possess a two component NS2B/NS3 protease which cleaves viral precursor proteins. Whereas for the WNV protease two crystal structures in complex with an inhibitor have been solved recently, no such information is available for the DV protease. Here, we report the generation of a homology model of DV NS2B/NS3 protease. Since it is known from the related WNV protease that it adopts a distinct conformation in free and in inhibitor-complexed form, a special emphasis was given to the analysis of the protease flexibility. Therefore, several models of DV NS2B/NS3 protease complexed with the peptidic inhibitor (Bz-Nle(P4)-Lys(P3)-Arg(P2)-Arg(P1)-H) were generated. The first DV protease model (DV-1) was constructed using the available crystal structure of the apo DV NS2B/NS3 protease. The second model (DV-2) was built taking the WNV NS3/NS2B protease in the inhibitor-complexed form as the template structure. Molecular dynamics simulations which were carried out for the WNV crystal structures as well as for the DV models provided an understanding of the role of NS2B for maintaining the protease in the active conformation. It was also demonstrated that NS2B is not only important for maintaining NS3 in the active form, but is also essential for establishing the interaction between residues from the S2 pocket and the peptidic inhibitor. The DV NS2B/NS3 model in the productive conformation can now be used for structure-based design purposes.

The mechanism of photoinduced electron transfer (PET) from the aromatic amino acids (Trp32, Tyr35 and Trp106) to the excited flavin mononucleotide (FMN) in the wild type (WT) and four single amino acid substitution isomers (E13T, E13Q, W32A and W32Y) of FMN binding protein (FBP) from the Desulfovibrio vulgaris (Miyazaki F) were simultaneously analyzed (Method A) with the Marcus-Hush (MH) theory and Kakitani-Mataga (KM) theory using ultrafast fluorescence dynamics of these proteins. In addition, the PET mechanism of the WT, E13T and E13Q FBP systems (Method B) were also analyzed with both MH and KM theories. The KM theory could describe all of the experimental fluorescence decays better than the MH theory by both Methods A and B. The PET rates were found to largely depend on the electrostatic energies between photo-products, isoalloxazine (Iso) anion and the PET donor cations, and the other ionic groups, and hence on static dielectric constants. The dielectric constant (ε(0)(DA)) around the PET donors and acceptor was separately determined from those (ε(0)(j), j = WT, E13T, E13Q, W32Y and W32A) in the domain between the Iso anion or the donor cations and the other ionic groups in the proteins. The values of ε(0)(DA) were always lower than those of ε(0)(j), which is reasonable because no amino acid exists between the PET donors and acceptor in all systems. The values of the dielectric constants ε(0)(j) (j = WT, E13T and E13Q) were similar to those obtained previously from the analysis of the crystal structures and the average lifetimes of these FBP proteins. Energy gap law in the FBP systems was examined. An excellent parabolic function of the logarithms of the PET rates was obtained against the total free energy gap. The PET in these FBP isomers mostly took place in the so-called normal region, and partly in the inverted region.