High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile ß-trefoil lectin domain from the mushroom Laetiporus sulphureus.

In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL(150)) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL(150) behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47 Å resolution and also that of the [LSL(150):(lactose)ß, γ)] binary complex at 1.67 Å resolution. This complex reveals two lactose molecules bound to the ß and γ sites of LSL(150), respectively. Isothermal titration calorimetry indicates that LSL(150) binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL(150) exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL(150) (LSL(t)) to the N-terminal end of various recombinant proteins. All the designed LSL(150)-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL(150) as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL(150) tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications.

Structural models of DYNLL1 with interacting partners: African swine fever virus protein p54 and postsynaptic scaffolding protein gephyrin.

DYNLL1, the smallest dynein light chain, interacts with different cargos facilitating their cellular transport. Usually the sequence recognized in the targets is homologous to the GIQVD or the KXTQT motifs with a glutamine that is important for binding. Here we add two new examples of DYNLL1 targets that can be classified into these two groups: ASFV p54 and gephyrin. Using NMR we demonstrate the direct interaction between DYNLL1 and two peptides derived from their interacting sequences. We model the structure of both complexes and show that the overall binding mode is preserved as in other complexes despite differences at the residue-specific interactions.

African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.

Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.

Identification of novel cellular proteins that bind to the LC8 dynein light chain using a pepscan technique.

Dynein is a minus end-directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid-1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection.

Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus.

The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies. A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6x His were added in reverse primers used to amplify the corresponding cDNAs. The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl-beta-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni-nitrilotriacetic acid agarose column. This method yielded 20-40 mg of highly pure and very stable proteins per liter of cell culture. Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6x His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions.

Selective destabilization of acidic phospholipid bilayers performed by the hepatitis B virus fusion peptide.

A peptide corresponding to the N-terminal region of the S protein of hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously demonstrated to perform aggregation and destabilization of acidic liposome bilayers and to adopt a highly stable beta-sheet conformation in the presence of phospholipids. The changes in the lipid moiety produced by this peptide have been followed by fluorescence depolarization and electron microscopy. The later was employed to determine the size and shape of the peptide-vesicle complexes, showing the presence of highly aggregated and fused structures only when negatively charged liposomes were employed. 1,6-Diphenyl-1,3,5-hexatriene depolarization measurements showed that the interaction of the peptide with both negatively charged and zwitterionic liposomes was accompanied by a substantial reduction of the transition amplitude without affecting the temperature of the gel-to-liquid crystalline phase transition. These data are indicative of the peptide insertion inside the bilayer of both types of liposomes affecting the acyl chain order, though only the interaction with acidic phospholipids leads to aggregation and fusion. This preferential destabilization of the peptide towards negatively charged phospholipids can be ascribed to the electrostatic interactions between the peptide and the polar head groups, as monitored by 1-(4-(trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene fluorescence depolarization analysis.

The Akt kinase signals directly to endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

Fusogenic activity of hepadnavirus peptides corresponding to sequences downstream of the putative cleavage site.

Sequence homology between the amino-terminal region of the S protein of hepatitis B Virus (HBV) and known fusion peptides from retroviruses and paramyxoviruses led us to propose that this region might be equally involved in the initial infective steps of hepadnaviruses. In fact, we showed that a synthetic peptide corresponding to the N-terminus region of the S protein of HBV had membrane-interacting properties and was able to induce liposome fusion adopting an extended (beta-sheet) conformation (Rodríguez-Crespo et al., 1996, 1995). We describe herein studies on the interaction of peptides derived from the N-terminal region of the S protein of duck (DHBV: Met-Ser-Gly-Thr-Phe-Gly-Gly-Ile-Leu-Ala-Gly-Leu-Ile-Gly-Leu-Leu) and woodchuck hepatitis B viruses (WHV: Met-Ser-Pro-Ser-Ser-Leu-Leu-Gly-Leu-Leu-Ala-Gly-Leu-Gln-Val-Val) with liposomes. These peptides were able to induce to a different extent aggregation, lipid mixing, and leakage of internal aqueous contents from both neutral and negatively charged phospholipid vesicles in a concentration-dependent and pH-independent manner. Fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene-labeled vesicles indicated that both peptides become inserted into the hydrophobic core of the lipid bilayer. Circular dichroism studies indicated that the DHBV peptide adopts an extended conformation in the presence of lipids, whereas the WHV peptide displays a high content of alpha-helical conformation. Therefore, these results extend our previous findings obtained for human hepatitis B virus to other members of the hepadnavirus family and suggest that this region of the S protein is important in the initial steps of the infective cycle.

Mutation of the five conserved histidines in the endothelial nitric-oxide synthase hemoprotein domain. No evidence for a non-heme metal requirement for catalysis.

Five conserved histidine residues are found in the human endothelial nitric-oxide synthase (NOS) heme domain: His-420, His-421, and His-461 are close to the heme, whereas His-146 and His-214 are some distance away. To investigate whether the histidines form a non-heme iron-binding site, we have expressed the H146A, H214A, H420A, H421A, and H461A mutants. The H420A mutant could not be isolated, and the H146A and H421A mutants were inactive. The H214A mutant resembled the wild-type enzyme in all respects. The H461A mutant had a low-spin heme, but high concentrations of L-Arg and tetrahydrobiopterin led to partial recovery of activity. Laser atomic emission showed that the only significant metal in NOS other than calcium and iron is zinc. The activities of the NOS isoforms were not increased by incubation with Fe(2+), but were inhibited by high Fe(2+) or Zn(2+) concentrations. The histidine mutations altered the ability of the protein to dimerize and to bind heme. However, the protein metal content, the inability of exogenous Fe(2+) to increase catalytic activity, and the absence of evidence that the conserved histidines form a metal site provide no support for a catalytic role for a non-heme redox-active metal.

Nitric oxide synthases catalyze the activation of redox cycling and bioreductive anticancer agents.

Nitric oxide synthases (NOSs) play a crucial role in the control of blood flow, memory formation, and the immune response. These proteins can be structurally divided into oxygenase and reductase domains. The reductase domain shares a high degree of sequence homology with P450 reductase, which is thought to be the major enzyme responsible for the one-electron reduction of foreign compounds, including bioreductive antitumor agents currently undergoing clinical trials. In view of the structural similarities between NOS and P450 reductase, we investigated the capacity of NOS to reduce the hypoxic cytotoxin tirapazamine, the antitumor agent doxorubicin, and also the redox cycling compound menadione. All three isoforms exhibited high levels of activity toward these compounds. In the case of doxorubicin and menadione, the activity of NOS II was 5-10-fold higher than the other enzymes, whereas with tirapazamine, the activities were broadly similar. NOS-mediated metabolism of tirapazamine resulted in a large increase in plasmid DNA strand breaks, demonstrating that the reduction was a bioactivation process. In addition, tirapazamine inhibited NOS activity. Because nitric oxide is implicated in maintaining tumor vascular homeostasis, it is conceivable that tirapazamine could potentiate its own toxicity by increasing the degree of hypoxia. This study suggests that the NOSs could play a key role in the therapeutic effects of tirapazamine, particularly because NOS activity is markedly increased in several human tumors. In addition, the presence of NOS in the heart indicates that these enzymes may contribute to the cardiotoxicity of redox cycling drugs, such as doxorubicin.

AMP-activated protein kinase phosphorylation of endothelial NO synthase.

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.

Nitric oxide synthase structure and electron transfer.

The nitric oxide synthases (NOS), although unrelated to the cytochromes P450 in terms of sequence, exhibit spectroscopic and catalytic properties strongly reminiscent of those of the P450 system. One important difference is the requirement of the NOS enzymes for tetrahydrobiopterin. The biopterin cofactor is shown by chemical studies to bind close to pyrrole ring D of the prosthetic heme group, a position confirmed recently for inducible NOS and endothelial NOS by crystal structures. The only plausible role so far for the tetrahydrobiopterin is as a transient electron donor for the activation of molecular oxygen. NADPH-derived electrons are provided to the heme by the NOS flavin domain, but the biopterin may be required to provide an electron at a faster rate than that supported by the flavin groups. Chimeras in which the reductase domains of the isoforms have been exchanged indicate that the overall rate of catalytic turnover is directly governed by the ability of the flavin domain to deliver electrons. Electron transfer from the flavin to the heme domain, and within the flavin and heme domains, is thus a critical determinant of the catalytic turnover of NOS.

Binding of dynein light chain (PIN) to neuronal nitric oxide synthase in the absence of inhibition.

PIN, an 89-amino-acid polypeptide found in a rat hippocampal cDNA library using the yeast two-hybrid system and various neuronal nitric oxide synthase (nNOS) fragments as bait, was reported to be an inhibitor of nNOS (Science 274, 774-778, 1996). PIN reportedly inhibited nNOS selectively and did not interact with either the endothelial or inducible nitric oxide synthase isoforms. Inhibition was attributed to the ability of PIN to dissociate the catalytically active nNOS homodimer. PIN is a dynein light chain (J. Biol. Chem. 271, 19358-19366, 1996), which suggested that PIN may serve as an axonal transport protein for nNOS. We have synthesized a rat PIN cDNA by recursive polymerase chain reaction and have expressed the protein in Escherichia coli. Recombinant PIN is a folded dimeric, mostly alpha-helical protein with a single deeply buried tryptophan residue. We have also expressed and purified the nNOS fragment to which PIN reportedly binds (residues 163-245). This recombinant peptide has a disordered secondary structure. Gel-filtration experiments show that PIN binds to both the full-length nNOS and nNOS fragment. However, PIN neither inhibits nNOS activity nor dissociates the nNOS dimer into monomeric species. PIN thus possibly functions as a dynein light chain involved in nNOS axonal transport but is not an inhibitor of the enzyme. Our results agree with the proposal (Cell 82, 743-752, 1995) that the PIN recognition sequence in nNOS both lies outside the catalytic core and is not part of the monomer-monomer contact region.

Endothelial nitric oxide synthase: modulations of the distal heme site produced by progressive N-terminal deletions.

cDNAs coding for bovine endothelial nitric oxide synthase (eNOS) with N-terminal deletions of 52, 91, and 105 amino acids were constructed, and the proteins were expressed in Escherichia coli and purified by affinity chromatography. All three truncated proteins bind heme and exhibit the ferrous-CO absorption maximum at 444 nm characteristic of thiolate heme ligation. Deletion of the first 52 amino acids yields a fully active dimeric protein with the same spectroscopic properties as the wild-type. The myristoylation, palmitoylation, and polyproline domains of the enzyme located in the deleted region are therefore not required for full catalytic activity. The delta91 and delta105 proteins, which exhibit altered dimerization equilibria, retain 20 and 12%, respectively, of the maximal activity. Resonance Raman and UV-vis spectroscopy indicate that, in the absence of tetrahydrobiopterin (H4B) and l-Arg, the wild-type and delta52 proteins are predominantly five coordinate high spin, whereas the delta91 and delta105 proteins are six coordinate low spin. The delta91 and delta105 mutants bind H4B, as indicated by a concomitant decrease in the low-spin component of the UV-vis spectrum, but the binding of l-Arg is extremely slow ( approximately 15 min). Dithiothreitol readily coordinates as the sixth iron ligand in the delta91 and delta105 mutants but not in the delta52 or wild-type proteins. The dithiothreitol can be completely displaced by l-Arg but not by H4B. Resonance Raman comparison of wild-type eNOS and nNOS confirms that, in the absence of H4B and l-Arg, eNOS is primarily high spin whereas nNOS is predominantly six coordinate, low spin. The results indicate that Cys-101 is not critical for the binding of H4B and imply that some of the protein residues involved in dimer formation and in preservation of active site integrity are located, probably at the monomer-monomer interface, in the N-terminal end of the protein.

Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus.

The gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (EIAV) was cloned from EIAV infected serum, expressed in E. coli, and the resultant protein purified to electrophoretic homogeneity. The protein was expressed in a soluble form and was purified by conventional protein separation methods. When analyzed by SDS-PAGE, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kDa monomer. Recombinant p26 (rp26), therefore, does not contain any intermolecular disulfide bond. Gel filtration chromatography also indicated that the protein occurs as a monomer in solution. Labeling of free sulphydryl groups with [1-14C]iodoacetamide suggests that none of the three cysteine residues of rp26 is involved in intramolecular disulfide bonds. The circular dichroism spectrum of rp26 was consistent with the following assignment of secondary structure elements: 51% a-helix, 15% beta-turn, and 34% aperiodic. Fluorescencespectroscopy revealed that the three tryptophan residues in rp26 occupy two different environments. These data support the conclusion that the recombinant protein is folded into an ordered and probably native conformation. Immunoblotting and enzyme immunoassay with EIAV infected sera demonstrated that recombinant p26 protein may be useful for diagnostic purposes.

Active site topologies and cofactor-mediated conformational changes of nitric-oxide synthases.

The active site topologies of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric-oxide synthases heterologously expressed in Escherichia coli have been examined using three aryldiazene (Ar-N=NH) probes. The topological information derives from (a) the rate and extent of aryl-iron complex formation in the presence and absence of tetrahydrobiopterin (H4B), Ca2+-dependent calmodulin (CaM), and L-arginine, and (b) the N-phenylprotoporphyrin IX regioisomer ratios obtained upon migration of the phenyl of the phenyl-iron complex to the heme nitrogen atoms. The N-phenylprotoporphyrin ratios indicate that the three NOS isoforms have related active site topologies with unencumbered space above all four pyrrole rings but particularly above pyrrole ring D. H4B binds directly above the heme pyrrole ring D or causes a conformational change that constricts that region, because H4B markedly decreases phenyl migration to pyrrole ring D. Small CaM-dependent changes in the nNOS N-phenylporphyrin isomer pattern are consistent with a conformational link between the CaM and heme sites in this protein. The ceiling height directly above the heme iron atom differs among the isoforms and is lower than in the P450 enzymes because only nNOS and iNOS react with 2-naphthyldiazene, and none of the isoforms reacts with p-biphenyldiazene. L-Arg blocks access to the heme iron atom in all three NOS isoforms and nearly suppresses the phenyldiazene reaction. The data indicate that topological differences, including differences in the size of the active site, are superimposed on the structural similarities among the NOS active sites.

Human endothelial nitric oxide synthase: expression in Escherichia coli, coexpression with calmodulin, and characterization.

Human endothelial nitric oxide synthase (eNOS) has been cloned and expressed in Escherichia coli. The spectroscopic properties and specific activity (100-130 nmol x min(-1) x mg(-1) at 37 degrees C) of the recombinant protein are similar to those of the bovine enzyme. FPLC and low-temperature SDS-PAGE indicate that the protein is mostly dimeric in both the absence and presence of tetrahydrobiopterin. Human eNOS thus has a higher tendency to dimerize than the bovine enzyme. A chloramphenicol-resistant, trc promoter-based plasmid has been constructed that allows coexpression of human calmodulin (CaM). Coexpression of CaM increases more than threefold the amount of expressed eNOS, stabilizes the recombinant protein, and significantly augments its specific activity (to 140-170 nmol x min(-1) x mg(-1) at 37 degrees C). The cytochrome c reduction activity is also improved by CaM coexpression. These increases in activity are not achieved by the addition of CaM to eNOS expressed in the absence of CaM. Gel filtration studies suggest that CaM coexpression produces a more elongated eNOS structure and alters the NADPH binding domain. CaM coexpression has been shown previously to be required for successful expression of the inducible NOS isoform, but this is the first demonstration that CaM coexpression improves the expression of a constitutive isoform.

Structural properties of the putative fusion peptide of hepatitis B virus upon interaction with phospholipids. Circular dichroism and Fourier-transform infrared spectroscopy studies.

A peptide corresponding to the N-terminal sequence of the S protein from hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously shown to interact with phospholipids and promote vesicle aggregation, phospholipid mixing, and liposome leakage, as well as erythrocyte lysis [Rodríguez-Crespo, I., Núñez, E., Gómez-Gutiérrez, J., Yélamos, B., Albar, J. P., Peterson, D. L. & Gavilanes, F. (1995) J. Gen. Virol. 76, 301-308]. The conformation of this putative fusion peptide has been studied, both at low and high peptide concentrations, by means of circular dichroism and Fourier-transform infrared spectroscopy, respectively. When the peptide is dissolved in trifluoroethanol, a significant population of alpha-helical structure is found in spite of the proline residue at position 11. In contrast, this hydrophobic oligopeptide has a high tendency to form large beta-sheet aggregates in aqueous buffers. Most of these aggregates can be eliminated by centrifugation. The peptide remaining in the supernatant adopts a non-ordered conformation. The aggregates can be dissociated by the anionic detergent sodium cholate, but the peptide still maintains an extended conformation. In the presence of acidic phospholipid vesicles, the putative fusion peptide adopts a highly stable beta-sheet conformation. Thus, unlike the fusion peptides of other viruses, an extended conformation seems to be the preferred structure when interacting with phospholipids. Such a conformation should be responsible for its membrane destabilization properties.

Endothelial nitric-oxide synthase. Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation.

Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH4). In the presence of BH4, eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added. The low-spin component of the spectrum of the BH4-free protein is decreased by the addition of BH4 without a corresponding increase in the high-spin component. Addition of BH4 decreases the low-spin population of eNOS even in the presence of excess L-arginine. These results indicate that BH4 directly modulates the heme environment. BH4-free eNOS is completely inactive, but catalytic activity is recovered when BH4 (EC50 approximately 200 nM) is added. The spectroscopically determined binding constants for L-arginine are approximately 1.9 microM in the presence and approximately 4.0 microM in the absence of BH4. The BH4-supplemented enzyme has an activity of 90-120 nmol of citrulline.min-1.mg-1 and Km values of 3 and 14 microM for L-arginine and N-hydroxy-L-arginine, respectively. Of particular interest is the finding by SDS-polyacrylamide gel electrophoresis that BH4-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH4-reconstituted protein. Addition of BH4, increases the percent of the dimer by only approximately 5%. The results establish that BH4 influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation.

Antigenicity of hepatitis B surface antigen proteins reconstituted with phospholipids.

Hepatitis B surface antigen (HBsAg) has been reconstituted with different phospholipid classes. All epitopes defined by a panel of monoclonal antibodies which recognize both group- and subtype-specific antigenic determinants showed specificity for acidic phospholipids. Electrostatic interactions between HBsAg proteins and acidic phospholipids are partly responsible for the complete recovery of the antigenic properties. In addition to the nature of the polar head group, the fatty acid composition of the phospholipid also influenced the recovery of the antigenic activity. Negatively charged phospholipids must bear at least one unsaturated fatty acid in order to be effective in recovering full antigenic activity of HBsAg. The results reported herein support the conclusion that the antigenic activity is dependent on the physical state of the phospholipid moiety. The appropriate membrane fluidity is required for optimum conformation but, once this conformation is established, additional interactions imparted by the various phospholipids give a difference in the patterns of antigenicity. The analysis of binding of the monoclonal antibodies allowed the classification of the epitopes into two groups according to their dependence on the lipid moiety. Of all the antigenic determinants only those close to the lipid-protein interface would change upon direct interaction with the phospholipids. The rest would depend on the correct protein conformation determined by the appropriate phospholipid composition.