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The kinetics of interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme domains in a rat neuronal nitric oxide synthesis (nNOS) holoenzyme in the presence and absence of added calmodulin using laser flash photolysis of CO dissociation in comparative studies on partially reduced NOS and a single domain NOS oxygenase construct is reported. The results attribute the slower IET between FMN and heme in the holoenzyme to the additional step of dissociation of the FMN domain from the reductase complex before reassociation with the oxygenase domain to form the electron-transfer competent output state complex.

[SDH.sup.Y236F] is described in terms of its constitution, enzyme activity, electrochemically driven catalysis, intramolecular electron transfer and spectroscopic properties. As a result of substitution, the hydrogen bonding network adjoining the active site is disturbed, resulting in an increased mobility of nearby arginine.

The kinetics of intersubunit intraprotein electronic transfer (IET) between the flavin monoclueotide (FMN) and heme domains in the nNOS (neuronal) oxyFMN (flavin mononucleotide) construct in the presence and absence of added calmodulin (CaM) were determined using laser flash photolysis of CO dissociation in comparative studies. The IET rate constant in the presence of CaM [262 [s.sup.-1]] was increased approximately 10-fold compared to that in the absence of CaM [22 [s.sup.-1]].

The two nonstoichiometric ligand binding sites are examined using plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the structural changes of the receptor to be monitored. The results reveal that these two binding sites are independent and non-interconvertible on the time scale of these experiments.

A novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS) are designed and expressed. Laser flash photolysis is used to investigate the kinetics of the intramolecular electron transfer (IET) between the FMN and the heme domains in a novel two-domain murine inducible iNOS oxyFMN construct in which only the FMN and oxygenase domains are expressed.

Mutations G473D and A208D were identified in patients with isolated sulphide oxidase (SO) deficiency, and the equivalent amino acids are localized to the vicinity of the molybdopterin active site in the X-ray structure of chicken SO. The results clearly implicate the importance go G473 and A208 in controlling the rates of the IET process in human SO and allow the deleterious impact of these mutations to be rationalized.

Here we report, for the first time, the formation of a biomimetic covalently imprinted polymeric sensor for a target ligand, the [delta]-opioid G-protein coupled receptor agonist DPDPE, which reproducibly exhibits subpicomolar binding affinity in an aqueous environment. In addition to having a well-defined and homogeneous binding site, the imprinted polymer template is quite stable to storage in both the dry and wet states and has at least 6 orders of magnitude higher affinities than exhibited by similar peptide-based molecular-imprinted polymers (MIPs) thus far. A highly sensitive optical detection methodology, plasmon-waveguide resonance spectroscopy, was employed, capable of measuring binding in real time and discriminating between ligand molecules, without requiring labeling protocols (fluorophores or radioisotopes). The DPDPE-imprinted polymer showed a broad structure--activity relationship profile, not unlike that found for protein receptors. Such sensitivity and robustness of MIPs suggests potential applications ranging from biowarfare agent detection to pharmaceutical screening.

Plasmon-waveguide resonance (PWR) spectroscopy can be applied to integral membrane proteins incorporated into a supported lipid bilayer without the need for labeling. With high sensitivity and wide dynamic range, this technique can be used to characterize the kinetics and thermodynamics of conformational events associated with the binding of ligands to G-protein-coupled receptors (GPCRs), and to directly examine the interactions of GPCRs with G proteins and other downstream effectors in signal transduction. This allows an easy distinction to be made between agonists, antagonists and inverse agonists, and provides a powerful new tool for studying membrane signaling and for drug development.

Results show that in the wild type Ectothiorhodospira halophila, the proton uptake by the chromophore of the photoactive yellow protein is from the external medium and not from an internal donor amino acid as shown by the mutational analyses. Further, the proton release is synchronized with the decay of the blue-shift state.

Research has been conducted on the intramolecular electron transfer. The study of the intramolecular electron transfer in sulfite oxidase has been carried out via the use of laser flash photolysis and the effect of viscosity on intramolecular electron transfer in proteins has been investigated.

Research points out that in the putidaredoxin-P450cam redox couple the complex fomation occurs electrostatically whereas in case putidaredoxin-NADH-putidaredoxin reductase, the association is driven by nonelectrostatic interactions as shown by laser flash photolysis technique.

Research demonstrates the importance of Glu139 substitution in the ferredoxin:NADP+ reductase in terms of efficiency of the negative charge driven electron transport with ferredoxin. Data further show that at low ionic strengths, the proteins in the complex are less optimal to the point of being unreactive.

X-ray structure of the photoactive blue-light receptor yellow protein reveals that its chromophore is buried in a hydrophobic core and tethered to the protein by means of hydrogen bonds. The hydrogen-bonding interactions between specific amino acid residues of the protein and the chromophore suggest that the residues are important for chromophore function.

Glu46Asp and Glu46Ala mutants were generated in photoactive yellow protein (PYP) to determine the effects of decreasing the length of the carbon chain and removing the carboxyl in the side chain on PYP's spectral and photocycle properties. Results indicate that the steady-state spectra of the dark-adapted states of the Glu46Asp and Glu46Ala mutants have radically blue-shifted species at neutral hydrogen-ion concentration. It was also noted that both mutants are photoactive, but demonstrate different kinetic properties.

Surface plasmon resonance (SPR) has become a popular method for investigating biomolecular interactions. A new variant of this technique, coupled plasmon-waveguide resonance (CPWR) spectroscopy, allows the characterization of anisotropic biological membranes. Plasmon resonance can therefore be used to study the molecular events involved in a wide variety of membrane processes, including energy conversion and signal transduction.