Phe1395 stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe 1395 was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP ؉ binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP ؉ with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP ؉ dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP ؉ -bound state as compared with wild type. We conclude that Phe 1395 regulates nNOSr catalysis in two ways. It facilitates NADP ؉ release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer. Nitric-oxide synthases (NOS)1 are homodimeric enzymes that synthesize NO via oxidation of L-Arg and participate in various physiological and pathological settings (1-6). In the NOS polypeptide, an N-terminal oxygenase domain is linked to a C-terminal reductase domain by a calmodulin (CaM)-binding sequence. The NOS oxygenase domain contains binding sites for iron protoporphyrin IX (heme), (6R)-5, 6, 7, 8-tetrahydro-Lbiopterin (H 4 B), and L-Arg and is the site where oxidative catalysis takes place. The NOS reductase domain (NOSr) contains binding sites for FMN, FAD, and NADPH and functions to transfer reducing equivalents from NADPH to the oxygenase domain.NOSr belongs to a small family of structurally related dualflavin reductases that also includes cytochrome P450 reductase (CYPR) (7, 8), methionine synthase reductase (9), and novel reductase-1 (10). These proteins are comprised of separate FMN and FAD/NADPH modules attached by a flexible hinge region (11,12). It is believed that these reductases are the product of gene fusion because their FMN and FAD/NADPH subdomains show a high similarity to flavodoxins (13) and ferredoxin NADP ϩ reductases (FNR) (14), respectively. In NOSr and related flavoproteins, the FAD receives electrons from NADPH via hydride transfer and then sequentially passes the electrons to the FMN cofactor. Ultimately, it is the 2-electron reduced FMN hydroqu...

Interactions among several components of the flagellar export apparatus of Salmonella were studied using affinity chromatography, affinity blotting, and fluorescence resonance energy transfer (FRET). The components examined were two integral membrane proteins, FlhA and FlhB, and two soluble components, FliH and the ATPase FliI. Affinity chromatography and affinity blotting demonstrated a heterologous interaction between FlhA and FlhB but not homologous FlhA-FlhA or FlhB-FlhB interactions. Both FlhA and FlhB consist of N-terminal transmembrane domains and C-terminal cytoplasmic domains (FlhA(C) and FlhB(C)). To study the interactions among the cytoplasmic components (FlhA(C), FlhB(C), FliH, and FliI), FRET measurements were carried out using fluorescein-5-isothiocyanate (FITC) as donor and tetramethylrhodamine-5- (and 6-) isothiocyanate (TRITC) as acceptor. To reveal the nature of the binding interactions, measurements were carried out in physiological buffer, at high salt (0.5 M NaCl) and in 30% 2-propanol. The results indicated that FlhA(C) could bind to FlhB(C) and both FlhA(C) and FlhB(C) could bind to themselves. Both FlhA(C) and FlhB(C) bound to FliH and FliI. Several in-frame deletion mutants of FliH were examined and found to have only minor effects of decreased binding to FlhA(C) and FlhB(C); deletions in the interior of the FliH sequence had a greater effect than those at the N terminus. The FliI mutants examined bound FlhA(C) and FlhB(C) about the same as or slightly more weakly than wild-type FliI. FliH bound more weakly to FliI carrying the N-terminal double mutation R7C/L12P than it did to wild-type FliI, confirming the importance of the N terminus of FliI for its interaction with FliH.

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Detergent (pentaoxyethylene octyl ether, C(8)E(5))-induced conformational changes of Humicola lanuginosa lipase (HLL) were investigated by stationary and time-resolved fluorescence intensity and anisotropy measurements. Activation of HLL is characterized by opening of a surface loop (the "lid") residing directly over the enzyme active site. The interaction of HLL with C(8)E(5) increases fluorescence intensities, prolongs fluorescence lifetimes, and decreases the values of steady-state anisotropy, residual anisotropy, and the short rotational correlation time. Based on these data, we propose the following model. Already below critical micellar concentration (CMC) the detergent can intercalate into the active site accommodating cleft, while the lid remains closed. Occupation of the cleft by C(8)E(5) also blocks the entry of the monomeric substrate, and inhibition of catalytic activity at [C(8)E(5)] less than or equal to CMC is evident. At a threshold concentration close to CMC the cooperativity of the hydrophobicity-driven binding of C(8)E(5) to the lipase increases because of an increase in the number of C(8)E(5) molecules present in the premicellar nucleates on the hydrophobic surface of HLL. These aggregates contacting the lipase should have long enough residence times to allow the lid to open completely and expose the hydrophobic cleft. Concomitantly, the cleft becomes filled with C(8)E(5) and the "open" conformation of HLL becomes stable.

We have characterized the relative efficacies of a number of protein crosslinking agents that have the potential for use in the crosslinking of proteinaceous matrices both in vitro and in vivo. The crosslinkers tested were; l-threose (LT), Genipin (GP), Methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), proanthrocyanidin (PA) and glutaraldehyde (GA). The relative effectiveness of the crosslinkers with regard to their saturating concentrations was: GA > PA > EDC > MG = GP >> LT. Most of the crosslinkers displayed a pH dependence and were more effective at more alkaline pH. At optimal pH and saturating conditions, the relative reaction rates of the crosslinkers were: PA = GA > EDC > GP > MG >> LT.

Amine-based CO 2 postcombustion capture is a commercialized technology for recovery CO 2 from flue gas, and the energy consumption is concentrated in the regenerative stage. The phase-change solvent system, monoethanolamine (MEA, absorbent) + solvent A (SA, solvent) + water (solvent), was investigated on the performances of CO 2 absorption and desorption. CO 2 -rich solvent can be continuously separated for the feeding flow of the regeneration tower; thereby, it significantly reduces the energy of regeneration. The results show that the solvent with suitable composition can easily separate the CO 2 -rich phase from the solvent, and the CO 2 -rich phase accounts for more than 98% of the total CO 2 . At 293−333 K, the temperature has no significant effect on the absorption rate and phase equilibrium. The estimation of energy consumption shows that the reboiler work can be reduced by 43.6% compared with that using 20 wt % MEA aqueous solution.

Study Design-Biochemical studies aimed at optimization of protein crosslinking formulations for the treatment of degenerative disc disease and subsequent biomechanical testing of tissues treated with these formulations.Objectives-To optimize protein crosslinking formulations for treatment of degenerating spinal discs.Summary of Background Data-Non-surgical exogenous crosslinking therapy is a potential new, non-invasive technology for the treatment of degenerative disc disease. The technology is based upon the injection of protein crosslinking reagents into the pathological disc to restore its mechanical properties and also to potentially increase the permeability of the tissue and so facilitate the exchange of waste products and nutrients.Methods-Diffusion of genipin was monitored following injection into spinal discs and the effects of surfactants on diffusion studied. Formulations for genipin and methylglyoxal were biochemically optimized and used to treat bovine spinal discs. Their effects on bovine annulus tissue were evaluated using a circumferential tensile test, while the genipin formulation was also tested with respect to its ability to reduce disc bulge under load.Results-Genipin exhibited a distinct time-dependent diffusion and sodium-dodecyl-sulfate, but not Tween-20, enhanced diffusion by 30%. Two crosslinkers, genipin and methylglyoxal, were inhibited by amines but enhanced by phosphate ions. Both formulations could enhance a number of physical parameters of bovine annulus tissue, while the genipin formulation could reduce disc bulge following injections into spinal discs.Conclusions-Formulations lacking amines and containing phosphate ions appear to be promising candidates for clinical use of the crosslinkers genipin and methylglyoxal.

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