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The results from the molecular dynamics simulations of nitrophorin 4 (NP4) at pH 5 and pH 7, modeled by selective deprotonation of acidic groups reveal that release rate of diatomic molecules from heme proteins could be varied by several orders of magnitude through modest adjustments in geminate rebinding and gating behavior.

The ferrous [Fe(super 2+)] complexes of nitrophorin 4 (NP4) with nitric oxide (NO), CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy are reported. The ferrous complexes exhibit absorption spectra that are similar to the analogous derivatives of myoglobin and exhibit heme coordination geometry that is consistent with a reduced form of the protein.

The study found that when a 45-bp DNA with a single CpG/TpG mispair was incubated with the cell extract and ATP, it was incised immediately 5' and 3' to the mismatched T. It was noted that the actual 5' and 3'-labeled fragments were similar to the cleaved products of thiamine DNA glycosylase.

The findings demonstrate that the pH-dependency for NO release is linked to loop dynamics and that solvent reorganization is apparently rate-limiting for formation of the initial iron-nitrsyl bond. The fact that NO diffusion out of the distal pocket is facilitated in proteins with destabilized loop conformations suggest that binding site loop opening does indeed regulate the release of NO.

Results show that nitrophorin 4 complexed with nitric oxide show ruffling of the heme in the presence or absence of nitric oxide. Data indicate that this protein-assisted heme ruffling may give rise to an autoreduction resistant nitrosyl-heme complex.

Evidence for stabilization of a nitric oxide-ferriheme complex via a ligand-induced conformational trap has been found. Kinetics and equilibria in ligand binding by nitrophorin (NP) 1-4 are discussed. Results of a study using kinetic and thermodynamic characterizations of NO and histamine binding to recombinant forms of all four NPs, suggest that tighter NO binding in the nitrophorins is the result of the trapping of the molecule in a nonpolar distal pocket, not of formation of especially strong hydrogen or hydrogen bonds. NPs 1-4 are ferriheme proteins from Rhodnius prolixus, a blood-sucking insect. They move NO to the victim, sequester histamine and inhibit coagulation of blood.

A study was conducted to analyze the crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex. Recombinant rat hepatoma TS was determined from an overexpressing Escherichia coli. Experimental results indicated that the rat protein undergoes a ligand-induced conformation change. Findings also showed an open conformation in the structures that may be correlated with the accumulation of various destabilizing factors.

The mechanism of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase was investigated. A functionally reactive chloroacetyl group was substituted for the propargyl group to examine whether the resulting analogue of CB3717 could become an irreversible inhibitor of thymidylate synthase. Results show that a sulfhydryl reactive residue directed to the folate binding site of thymidylate synthase may diffuse to the active site cysteine and form a covalent bond with the compound.

Research into the possibility of a physiological function for the dimeric form of thioredoxin indicates that there are a number of possible functions unique to the dimer form. These include a mechanism for the active-site in tissues or a protective mechanism for the active-site of the protein during secretion. Thioredoxin is an element of the early pregnancy factor system. The activity is not affected by the mutation of active-site residues, but mutation of Cys 73 to Ser eliminates it completely.

The structure of thymidylate synthase.5-fluoro-dUMP.CH2THF (TS.FdUMP.CH2THF) was analyzed to characterize the enzyme kinetics of Escherichia coli TS. Structural analysis of the covalent ternary complex indicated the stepwise mechanism of TS eliminations involving the abstraction of C5H. The Cys 146 sulfur adduct also remained axial to the nucleotide ring during catalysis and the enzyme utilized strain and electrostatic interactions to polarize or break the sulfur adduct to C6 during catalysis.

The expression and purification of milligram quantities of nitrophorin I and the rate and equilibrium binding constants for nitric oxide dissociation from protein were analyzed under varied hydrogen-ion concentrations (pH). The binding of nitrophorin I isolated from the salivary glands of Rhodnius prolixus to nitric oxide was affected by changes in pH. Furthermore, NO binding to nitrophorin I was hindered by acidic conditions due to pH-dependent alterations in protein conformation.

Molecular replacement studies of a T4 phage enzyme thymidylate synthase (T4TS) reveals the crystal structures to be open and unliganded, with three inserts and one deletion in the coding region. One side of the enzyme surface contains all these sites, which encode the molecular interactions. Folyl glutamate binds more strongly to T4TS than to Escherichia coli TS.

We have found CueO from Escherichia coli to have a robust cuprous oxidase activity, severalfold higher than any homologue. These data suggest that a functional role for CueO in protecting against copper toxicity in vivo includes the removal of Cu(I).

Heme‐nitric oxide/oxygen binding (H‐NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H‐NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H‐NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H‐NOX and coiled‐coil domains. Here, we report the full NMR structure for CO‐ligated Sw H‐NOX in the presence and absence of stimulator compound IWP‐051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi‐empirical quantum potential energy approach. Although IWP‐051 binding is weak, a single binding conformation was found at the interface of the two H‐NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix‐connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure‐based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H‐NOX proteins. The largest dynamical loop lies at the interface between Sw H‐NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction. PDB Code(s): 6OCV and 6WQE; [ABSTRACT FROM AUTHOR]

The rebinding kinetics of NO and CO to nitrophorin 4 (NP4) are examined as a function of iron oxidation state and the acidity of the environment. The analysis of the ultrafast kinetics has shown that the equilibrium off-rate of NO is proportional to the open state population as well as the pH-dependent amplitude of escape from the open pocket.

The crystal structure characterization of Vibrio harveyi, bacterial luciferase bound to flavin mononucleotide (FMN) at a resolution of 2.3 [Angstrom] is reported. The results provide insight into the functional role of [beta] subunit of the luciferase heterodimer that stabilizes the active conformation of the [alpha] subunit.

Site-directed mutants of three major nitrophorins, NP2, NP1 and NP4, are prepared in order to examine the stabilization of the ferric-NO heme center and preservation of reversible binding that has facilitated these proteins' NO storage, transport and release functions. The carboxyls have shown a significant role stabilizing [Fe.sup.III]-NO over [Fe.sup.II]-NO and the p[K.sub.a]s of the carboxyl groups has varied significantly, but all are largely deprotonated at pH 7.5 except E124.

The studies have shown that by direct observation of a geminate recombination phase for CO photolysis, the binding of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole) or BAY 41-2272 has altered the heme distal pocket. A powerful tool is provided for examining NO-independent stimulation of soluble guanylyl/guanylate cyclase (sGC) and the discovery of improved compounds for the treatment of cardiovascular disease.

The low-frequency mode activity of metalloporphyrins is analyzed for iron porphine-halides and nitrophorin 4 (NP4) by using femtosecond coherence spectroscopy (FCS) in combination with polarized resonance Raman spectroscopy and density functional theory (DFT). The results have shown the potential of FCS as a sensitive probe of dynamic and functionally important nonplanar heme vibrational excitations that are induced by the protein environmental forces or by the chemical reactions in the aqueous phase.

The X-ray crystal structure of GTP cyclohydrolase (GCH) III from Methanocaldococcus jannaschii (MJ0145) bound to its substrate GTP is determined. The arrangement of metal ions in the active site has suggested that GCH III has used two metal ion catalysis and the structure of GCH III has extended the repertoire of the reactions with a palm fold to include cyclohydrolase chemistry.

The crystal structure of the cyan-fluorescent Cerulean green fluorescent protein (GFP), which is a variant of enhanced cyan fluorescent protein (ECFP), is determined. The results have shown that the high-resolution X-ray structure of Cerulean GFP is consistent with a single ground state conformation for the chromophore and its protein environment, although the broad absorbance doublet is persisting in the crystal.

The ferriheme resonances of low-spin nitrophorins (NP) 1 and 4 were assigned and compared to those of NP2 by using NMR spectroscopy. The effective nodal plane of low-spin NP1, NP4, and NP2 complexes in all cases of imidazole and histamine complexes is found to be quite similar to the average of the His-59 or -57 and the exogenous ligand angles seen in the X-ray crystal structures.

The first view of a transnitrosated protein, human thioredoxin-1-S-Nitrosated (hTrx-SNO) is provided, which displays an unexpected buried SNO moiety. A new mechanism for transnitrosation reactions is suggested that further solidifies a role for human thioredoxin in SNO signaling and metabolism.

The crystal structure of apo-CusF, a periplasmic protein involved in copper and silver resistance, is used as a unique topology for a copper-binding protein. It is observed that the growing health concern of antibiotic-resistant micro-organism need more investigation into the metal resistance systems and their relation with other antibiotic resistance systems.

The conformational change and mobility in nitrophorin 4 as a function of ph and NO binding is presented. The striking features of both the 'open' and 'closed' conformers exist under all conditions, suggesting that the flexible loops can transition with relative ease between conformational states.

How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC‐1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N‐terminus in sGC activates cyclase activity near the C‐terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small‐molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled‐coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad‐repeating pattern for coiled‐coil motifs, but also invariant positions that disfavor coiled‐coil stability. Full‐length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled‐coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction. [ABSTRACT FROM AUTHOR]

The resonance Raman spectra of nitrophorin 1, obtained from the saliva of Rhonius prolixus, a blood sucking insect, is done in the absence and presence of nitric oxide and in the presence of cyano adducts. The results obtained are used to clarify the ligand geometry in the nitrophorins.

Significance: Soluble guanylyl/guanylate cyclase (sGC) is the primary receptor for nitric oxide (NO) and is central to the physiology of blood pressure regulation, wound healing, memory formation, and other key physiological activities. sGC is increasingly implicated in disease and is targeted by novel therapeutic compounds. The protein displays a rich evolutionary history and a fascinating signal transduction mechanism, with NO binding to an N-terminal heme-containing domain, which activates the C-terminal cyclase domains. Recent Advances: Crystal structures of individual sGC domains or their bacterial homologues coupled with small-angle x-ray scattering, electron microscopy, chemical cross-linking, and Förster resonance energy transfer measurements are yielding insight into the overall structure for sGC, which is elongated and likely quite dynamic. Transient kinetic measurements reveal a role for individual domains in lowering NO affinity for heme. New sGC stimulatory drugs are now in the clinic and appear to function through binding near or directly to the sGC heme domain, relieving inhibitory contacts with other domains. New sGC-activating drugs show promise for recovering oxidized sGC in diseases with high inflammation by replacing lost heme. Critical Issues: Despite the many recent advances, sGC regulation, NO activation, and mechanisms of drug binding remain unclear. Here, we describe the molecular evolution of sGC, new molecular models, and the linked equilibria between sGC NO binding, drug binding, and catalytic activity. Future Directions: Recent results and ongoing studies lay the foundation for a complete understanding of structure and mechanism, and they open the door for new drug discovery targeting sGC. Antioxid. Redox Signal. 26, 107-121. [ABSTRACT FROM AUTHOR]

Atomic Structure of Thymidylate Synthase: Target for Rational Drug Design THYMIDYLATE SYNTHASE (TS) (E.C. 2.1.1.45) CATALYZESthe conversion of deoxyuridine monophosphate (dUMP) and 5,10-methylenetetrahydrofolate (CH2H4folate) to deoxythymidine monophosphate (dTMP) and dihydrofolate. [...]