Membrane binding motif of the P-type cardiotoxin

Carditoxins (CTXs) from cobra snake venoms, the basic 60-62 residue all-beta sheet polypeptides, are known to bind to and impair the function of cell membranes. To assess the membrane induced conformation and orientation of CTXs, the interaction of the P-type cardiotoxin II from Naja oxiana snake venom (CTII) with perdeuterated dodecylphosphocholine (DPC) was studied using ( 1 )H-NMR spectroscopy and diffusion measurements. Under conditions where the toxin formed a well-defined complex with DPC, the spatial structure of CTII with respect to the presence of tightly bound water molecules in loop II, was calculated using the torsion angle dynamics program DYANA. The structure was found to be similar, except for subtle changes in the tips of all three loops, to the previously described "major" form of CTII in aqueous solution illustrated by the "trans" configuration of the Val7-Pro8 peptide bond. No "minor" form with the "cis" configuration of the above bond was found in the micelle-bound state. The broadening of the CTII backbone proton signals by 5, 16-doxylstearate relaxation probes, together with modeling based on the spatial structure of CTII, indicated a periphery mode of binding of the toxin molecule to the micelle and revealed its micelle interacting domain. The latter includes a hydrophobic region of CTII within the extremities of loops I and III (residues 5-11, 46-50), the basement of loop II (residues 24-29,31-37) and the belt of polar residues encircling these loops (lysines 4,5,12,23,50, serines 11,46, histidine 31, arginine 36). It is suggested that this structural motif and the mode of binding can be realized during interaction of CTXs with lipid and biological membranes.     

Interaction of the P-type cardiotoxin with phospholipid membranes.

The cardiotoxin (cytotoxin II, or CTII) isolated from cobra snake (Naja oxiana) venom is a 60-residue basic membrane-active protein featuring three-finger beta sheet fold. To assess possible modes of CTII/membrane interaction 31P- and 1H-NMR spectroscopy was used to study binding of the toxin and its effect onto multilamellar vesicles (MLV) composed of either zwitterionic or anionic phospholipid, dipalmitoylglycerophosphocholine (Pam2Gro-PCho) or dipalmitoylglycerophosphoglycerol (Pam2Gro-PGro), respectively. The analysis of 1H-NMR linewidths of the toxin and 31P-NMR spectral lineshapes of the phospholipid as a function of temperature, lipid-to-protein ratios, and pH values showed that at least three distinct modes of CTII interaction with membranes exist: (a) nonpenetrating mode; in the gel state of the negatively charged MLV the toxin is bound to the surface electrostatically; the binding to Pam2Gro-PCho membranes was not observed; (b) penetrating mode; hydrophobic interactions develop due to penetration of the toxin into Pam2Gro-PGro membranes in the liquid-crystalline state; it is presumed that in this mode CTII is located at the membrane/water interface deepening the side-chains of hydrophobic residues at the tips of the loops 1-3 down to the boundary between the glycerol and acyl regions of the bilayer; (c) the penetrating mode gives way to isotropic phase, stoichiometrically well-defined CTII/phospholipid complexes at CTII/lipid ratio exceeding a threshold value which was found to depend at physiological pH values upon ionization of the imidazole ring of His31. Biological implications of the observed modes of the toxin-membrane interactions are discussed.     

Heparin binding stabilizes the membrane-bound form of cobra cardiotoxin.

It has been shown previously that the long chain fragments of heparin bind to the beta-strand cationic belt of the three-finger cobra cardiotoxin (or cytotoxin, CTX) and hence enhance its penetration into phospholipid monolayer under physiological ionic conditions. By taking lysophosphatidylcholine (LPC) micelles as a membrane model, we have shown by (1)H NMR study that the binding of heparin-derived hexasaccharide (Hep-6) to CTX at the beta-strand region can induce conformational changes of CTX near its membrane binding loops and promote the binding activity of CTX toward LPC. The Fourier-transform infrared spectra and NMR nuclear Overhauser effect of Hep-6.CTX and CTX.LPC complex in aqueous buffer also supplemented the aforementioned observation. Thus, the detected conformational change may presumably be the result of structural coupling between the connecting loops and its beta-strands. This is the first documentation of results showing how the association of hydrophilic carbohydrate molecules with amphiphilic proteins can promote hydrophobic protein-lipid interaction via the stabilization of its membrane-bound form. A similar mechanism involving tripartite interactions of heparin, protein, and lipid molecules may be operative near the extracellular matrix of cell membranes.     

Docking studies on PARP-1 inhibitors: insights into the role of a binding pocket water molecule

The binding mode of a series of competitive PARP-1 inhibitors was investigated employing a molecular docking approach by using Autodock 3.0. A particular attention was given to the role played by a water molecule present in some but not all the so far available crystal structures of the catalytic domain of PARP-1. Good correlation between calculated binding energies and experimental inhibitory activities was obtained either by including (r2=0.87) or not (r2=0.84) the structural water molecule. Closer inspection of our results suggested that this water molecule should be considered part of the hydration shell of polar inhibitors and not as a structural water.     

Two forms of cytotoxin II (cardiotoxin) from Naja naja oxiana in aqueous solution: spatial structures with tightly bound water molecules.

1H-NMR spectroscopy data, such as NOE intraprotein and (bound water)/protein contacts, 3J coupling constants and deuterium exchange rates were used to determine the in-solution spatial structure of cytotoxin II from Naja naja oxiana snake venom (CTII). Exploiting information from two 1H-NMR spectral components, shown to be due to cis/trans isomerization of the Val7-Pro8 peptide bond, spatial structures of CTII minor and major forms (1 : 6) were calculated using the torsion angle dynamics algorithm of the DYANA program and then energy refined using the FANTOM program. Each form, major and minor, is represented by 20 resulting conformers, demonstrating mean backbone rmsd values of 0.51 and 0.71 A, respectively. Two forms of CTII preserve the structural skeleton as three large loops, including two beta-sheets with bend regions, and demonstrate structural differences at loop I, where cis/trans isomerization occurs. The CTII side-chain distribution constitutes hydrophilic and hydrophobic belts around the protein, alternating in the trend of the three main loops. Because of the Omega-shaped backbone, formed in participation with two bound water molecules, the tip of loop II bridges the tips of loops I and III. This ensures the continuity of the largest hydrophobic belt, formed with the residues of these tips. Comparison revealed pronounced differences in the spatial organization of the tips of the three main loops between CTII and previous structures of homologous cytotoxins (cardiotoxins) in solution.     

Putative implication of alpha-amylase loop 7 in the mechanism of substrate binding and reaction products release

Alpha-amylases are widespread endo-enzymes involved in the hydrolysis of internal alpha-(1,4) glycosidic linkages of starch polymers. Molecular modeling of amylose-amylase interactions is a step toward enzymatic mechanism understanding and rational design of new enzymes. From the crystallographic complex of barley alpha-amylase AMY2-acarbose, the static aspects of amylose-amylase docking have been characterized with a model of maltododecaose (DP12) (G. André, A. Buléon, R. Haser, and V. Tran, Biopolymers 1999, Vol. 50, pp. 751-762; G. André and V. Tran, Special Publication no. 246 1999, The Royal Society of Chemistry, H. J. Gilbert, G. J. Davies, B. Henrissat, and B. Svensson, Eds., Cambridge, pp. 165-174). These studies, consistent with the experimental subsite mapping (K. Bak-Jensen, G. André, V. Tran, and B. Svensson, Journal of Biological Chemistry, to be published), propose a propagation scheme for an amylose chain in the active cleft of AMY2. The topographical overview of alpha-amylases identified loop 7 as a conserved segment flanking the active site. Since some crystallographic experiments suspected its high flexibility, its putative motion was explored through a robotic scheme, an alternate route to dynamics simulations that consume CPU time. The present article describes the characteristics of the flexibility of loop 7: location and motion in AMY2. A back-and-forth motion with a large amplitude of more than 0.6 nm was evaluated. This movement could be triggered by two hinge residues. It results in the loop flipping over the active site to enhance the docking of the native helical substrate through specific interactions, it positions the catalytic residues, it distorts the substrate towards its transition state geometry, and finally monitors the release of the products after hydrolysis. The residues involved in the process are now rational mutation points in the hands of molecular biologists.     

Differential determinants for peptide and non-peptidyl ligand binding to the motilin receptor. Critical role of second extracellular loop for peptide binding and action.

The predicted second extracellular loop domain of the motilin receptor is of particular interest because it is a region that is quite distinct from the analogous regions in other family members that are most closely related and because the initial report of the photoaffinity labeling of a domain of this receptor included this region (Coulie, B. J., Matsuura, B., Dong, M., Hadac, E. M., Pinon, D. I., Feighner, S. D., Howard, A. D., and Miller, L. J. (2001) J. Biol. Chem. 276, 35518-35522). In the current work, motilin receptor constructs were prepared that included sequential deletions ranging from single residues to twelve amino acid segments throughout this 67 amino acid domain. Each construct was expressed in COS cells and characterized for motilin radioligand binding and motilin-stimulated intracellular calcium responses. The only segments that had negative impact on motilin binding and biological activity included deletion constructs DeltaCys(235), Delta179-182, and Delta241-246. Cys(235) is likely involved in the highly conserved and functionally important disulfide bond linking the first and second loops of G protein-coupled receptors. Alanine replacements for each of the amino acid residues in the other two segments revealed that the perimembranous residues at both ends of this loop, Val(179) and Leu(245) and Arg(246), were responsible for the negative impact on motilin binding and biological activity. Of note, these mutants responded normally to the non-peptidyl agonist, erythromycin. These data support important functional roles for both amino-terminal and carboxyl-terminal perimembranous regions of the second loop for responses to the natural agonist peptide, while supporting independent determinants for action of a non-peptidyl agonist ligand.     

The role of water in ligand binding

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Exploration of the complex modulatory role of water in ligand–target binding is a current challenge of drug design. This review reports on recent advances of prediction of water structure and function in the context of ligand engineering. The surveyed theoretical approaches showed remarkable progress in the past years. Beyond complementing experiments, they also supplied unmeasurable data. For example, thermodynamic calculations improved ligand binding by the selection of certain water molecules for structural replacement. Molecular dynamics and explicit solvent models remained indispensable to achieve precise results. Topographical analyses of hydration networks proved useful for the prediction of the stabilizing role of interconnected water clusters mediating target–ligand interactions.     

Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding.

The three-dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti-trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5'-AAT-3' region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen-bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen-bonded contact with an amidinium nitrogen atom, N3 of the 5' end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.     

Glycosphingolipid-facilitated membrane insertion and internalization of cobra cardiotoxin. The sulfatide.cardiotoxin complex structure in a membrane-like environment suggests a lipid-dependent cell-penetrating mechanism for membrane binding polypeptides

Cobra cardiotoxins, a family of basic polypeptides having lipid- and heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 angstroms resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.     

Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.     

Structural and dynamic characterization of omega-conotoxin MVIIA: the binding loop exhibits slow conformational exchange

omega-Conotoxin MVIIA is a 25-residue, disulfide-bridged polypeptide from the venom of the sea snail Conus magus that binds to neuronal N-type calcium channels. It forms a compact folded structure, presenting a loop between Cys8 and Cys15 that contains a set of residues critical for its binding. The loop does not have a unique defined structure, nor is it intrinsically flexible. Broadening of a subset of resonances in the NMR spectrum at low temperature, anomalous temperature dependence of the chemical shifts of some resonances, and exchange contributions to J(0) from (13)C relaxation measurements reveal that conformational exchange affects the residues in this loop. The effects of this exchange on the calculated structure of omega-conotoxin MVIIA are discussed. The exchange appears to be associated with a change in the conformation of the disulfide bridge Cys8-Cys20. The implications for the use of the omega-conotoxins as a scaffold for carrying other functions is discussed.     

The long and short flavodoxins: I. The role of the differentiating loop in apoflavodoxin structure and FMN binding

Flavodoxins are well known one-domain alpha/beta electron-transfer proteins that, according to the presence or absence of a approximately 20-residue loop splitting the fifth beta-strand of the central beta-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split beta-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see Lopez-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernandez-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184-47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones.     

Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the A ring of bound colchicine molecule

Isotypes of vertebrate tubulin have variable amino acid sequences, which are clustered at their C-terminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first-order conditions. Experiments with individual isotypes established that the presence of beta(III) in the purified tubulin is responsible for the biphasic kinetics. Because the isotypes mainly differ at the C termini, the colchicine-binding kinetics of unfractionated tubulin and the beta(III) isotype, cleaved at the C termini, have been tested under pseudo-first-order conditions. Removal of the C termini made no difference to the nature of the kinetics. Sequence alignment of different beta isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. To establish whether these differences lie at the colchicine-binding site or not, homology modeling of all beta-tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A ring of colchicine at the colchicine-binding site on beta tubulin. While the beta(III) isotype has two hydrophilic residues (serine(242) and threonine(317)), both beta(II) and beta(IV) have two hydrophobic residues (leucine(242) and alanine(317)). beta(II) has isoleucine at position 318, while beta(III) and beta(IV) have valine at that position. Thus, these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine-binding kinetics of the different isotypes of tubulin.     

Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding

We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ～10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

Acanthamoeba castellanii: characterization of an adhesin molecule.

Acanthamoeba castellanii is a free-living protozoan that causes keratitis in humans and has been associated with pneumonia and granulomatous amebic encephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In this study, several mouse monoclonal antibodies (MAb) generated to whole Acanthamoeba trophozoites identified surface membrane epitopes by ELISA and IFA. Nine antibodies inhibited adherence of [(35)S]-methionine-labeled Acanthamoeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium periodate treatment, but not proteinase K digestion, of whole Acanthamoeba destroyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7H6, suggesting that the adherence epitopes are carbohydrates. Other antibodies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the peptide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa band on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirming that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adherence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in soluble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibited from binding to whole trophozoites nor membrane extracts by mannose or mannan in competitive binding assays. When our Acanthamoeba membrane preparations were electrophoresed and immunoblotted with alpha-d-mannosylated-biotin albumin, no bands were recognized in the >207 kDa range by our adherence-associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein.     

Localization of bound water in the solution structure of the immunoglobulin binding domain of streptococcal protein G. Evidence for solvent-induced helical distortion in solution.

The presence of bound water in the solution structure of the IgG binding domain of streptococcal protein G has been investigated by nuclear magnetic resonance using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. The backbone amide protons of three residues, Ala20, Gln32 and Tyr33, are found to be in close proximity to bound water. Examination of the three-dimensional structure of the IgG binding domain indicates that in the vicinity of these three residues there are no backbone groups that do not already participate in hydrogen bonding and there are no suitably placed side-chain groups available for hydrogen bonding with water. As the lifetime of the bound water detected in this nuclear magnetic resonance experiment is greater than about one nanosecond, it is likely that the two bound water molecules participate in a bifurcating hydrogen bonding network comprising a CO-NH hydrogen bonded pair, such that the water molecule accepts a hydrogen bond from the NH proton and donates one to the carbonyl oxygen with the result that the amide proton is involved in a three center hydrogen bond. On the basis of the structure, one water molecule participates in such an interaction with the Ala20(NH)-Met1(CO) hydrogen bonded pair at the beginning of an anti-parallel beta-sheet, and the other with the Tyr33(NH)-Val29(CO) hydrogen bonded pair in the single alpha-helix. The latter, which is external and solvent accessible, is associated with a distortion in the alpha-helix centered around Tyr33 which consists of a significant increase in the CO(i-4)-N(i) and CO(i-4)-NH(i) distances relative to those in the rest of the helix, as well as a significant departure in the phi, psi angles of Tyr33 relative to regular helical geometry. Such solvent induced distortions in alpha-helices have been previously noticed in crystal structures and were postulated as possible folding intermediates for helical structures. The present observation of this phenomenon in solution indicates, however, that these water molecules are tightly bound and represent an integral part of the protein framework.     

Physiological role for the membrane bound ascorbate-TMPD oxidase in pseudomonas putida.

The activity of the membrane-bound ascorbate-TMPD oxidase in Pseudomonas putida varies with growth conditions and age of the culture. A comparison of the effects of cyanide and azide on the oxidation of various substrates suggests that ascorbate-TMPD oxidase is not the terminal oxidase for NADH or succinate oxidation. However, it does have a role in the oxidation of nicotinate, and may act as an additional terminal oxidase under certain other growth conditions.