Central Β-barrel Research Articles

Lateral gating mechanism and plasticity of the β-barrel assembly machinery complex in micelles and Escherichia coli.

The β-barrel assembly machinery (BAM) mediates the folding and insertion of the majority of outer membrane proteins (OMPs) in gram-negative bacteria. BAM is a penta-heterooligomeric complex consisting of the central β-barrel BamA and four interacting lipoproteins BamB, C, D, and E. The conformational switching of BamA between inward-open (IO) and lateral-open (LO) conformations is required for substrate recognition and folding. However, the mechanism for the lateral gating or how the structural details observed in vitro correspond with the cellular environment remains elusive. In this study, we addressed these questions by characterizing the conformational heterogeneity of BamAB, BamACDE, and BamABCDE complexes in detergent micelles and/or Escherichia coli using pulsed dipolar electron spin resonance spectroscopy (PDS). We show that the binding of BamB does not induce any visible changes in BamA, and the BamAB complex exists in the IO conformation. The BamCDE complex induces an IO to LO transition through a coordinated movement along the BamA barrel. However, the extracellular loop 6 (L6) is unaffected by the presence of lipoproteins and exhibits large segmental dynamics extending to the exit pore. PDS experiments with the BamABCDE complex in intact E. coli confirmed the dynamic behavior of both the lateral gate and the L6 in the native environment. Our results demonstrate that the BamCDE complex plays a key role in the function by regulating lateral gating in BamA.

Conformational Flexibility of the Protein Insertase BamA in the Native Asymmetric Bilayer Elucidated by ESR Spectroscopy.

The β‐barrel assembly machinery (BAM) consisting of the central β‐barrel BamA and four other lipoproteins mediates the folding of the majority of the outer membrane proteins. BamA is placed in an asymmetric bilayer and its lateral gate is suggested to be the functional hotspot. Here we used in situ pulsed electron‐electron double resonance spectroscopy to characterize BamA in the native outer membrane. In the detergent micelles, the data is consistent with mainly an inward‐open conformation of BamA. The native membrane considerably enhanced the conformational heterogeneity. The lateral gate and the extracellular loop 3 exist in an equilibrium between different conformations. The outer membrane provides a favorable environment for occupying multiple conformational states independent of the lipoproteins. Our results reveal a highly dynamic behavior of the lateral gate and other key structural elements and provide direct evidence for the conformational modulation of a membrane protein in situ.

Identification of Receptor Binding Proteins in Flagellotropic Agrobacterium Phage 7-7-1.

The rapid discovery of new and diverse bacteriophages has driven the innovation of approaches aimed at detailing interactions with their bacterial hosts. Previous studies on receptor binding proteins (RBPs) mainly relied on their identification in silico and are based on similarities to well-characterized systems. Thus, novel phage RBPs unlike those currently annotated in genomic and proteomic databases remain largely undiscovered. In this study, we employed a screen to identify RBPs in flagellotropic Agrobacterium phage 7-7-1. Flagellotropic phages utilize bacterial flagella as receptors. The screen identified three candidate RBPs, Gp4, Gp102, and Gp44. Homology modelling predicted that Gp4 is a trimeric, tail associated protein with a central β-barrel, while the structure and function of Gp102 and Gp44 are less obvious. Studies with purified Gp41-247 confirmed its ability to bind and interact with host cells, highlighting the robustness of the RBP screen. We also discovered that Gp41-247 inhibits the growth of host cells in a motility and lipopolysaccharide (LPS) dependent fashion. Hence, our results suggest interactions between Gp41-247, rotating flagellar filaments and host glycans to inhibit host cell growth, which presents an impactful and intriguing focus for future studies.

Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is required for the progression of the Plasmodium's life cycle and is therefore a promising malaria drug target. PfPKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays a crucial role in PfPKG regulation as it is the primary determinant for the inhibition and cGMP-dependent activation of the catalytic domain. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking. Here, we employed molecular dynamics simulations to model four key states along the thermodynamic cycle for the cGMP-dependent activation of the PfPKG CBD-D domain. The simulations were compared to NMR data, and they revealed that the PfPKG CBD-D activation pathway samples a compact intermediate in which the N- and C-terminal helices approach the central β-barrel. In addition, by comparing the cGMP-bound active and inactive states, the essential binding interactions that differentiate these states were identified. The identification of structural and dynamical features unique to the cGMP-bound inactive state provides a promising basis to design PfPKG-selective allosteric inhibitors as a viable treatment for malaria.

Structural characteristics of lipocalin allergens: Crystal structure of the immunogenic dog allergen Can f 6.

Lipocalins represent the most important protein family of the mammalian respiratory allergens. Four of the seven named dog allergens are lipocalins: Can f 1, Can f 2, Can f 4, and Can f 6. We present the structure of Can f 6 along with data on the biophysical and biological activity of this protein in comparison with other animal lipocalins. The Can f 6 structure displays the classic lipocalin calyx-shaped ligand binding cavity within a central β-barrel similar to other lipocalins. Despite low sequence identity between the different dog lipocalin proteins, there is a high degree of structural similarity. On the other hand, Can f 6 has a similar primary sequence to cat, horse, mouse lipocalins as well as a structure that may underlie their cross reactivity. Interestingly, the entrance to the ligand binding pocket is capped by a His instead of the usually seen Tyr that may help select its natural ligand binding partner. Our highly pure recombinant Can f 6 is able to bind to human IgE (hIgE) demonstrating biological antigenicity.

Structure and Reaction Mechanism of the LigJ Hydratase: An Enzyme Critical for the Bacterial Degradation of Lignin in the Protocatechuate 4,5-Cleavage Pathway.

LigJ from the soil bacterium Sphingobium sp. SYK-6 catalyzes the reversible hydration of (3 Z)-2-keto-4-carboxy-3-hexenedioate (KCH) to 4-carboxy-4-hydroxy-2-oxoadipate (CHA) in the degradation of lignin in the protocatechuate 4,5-cleavage pathway. LigJ is a member of the amidohydrolase superfamily and an enzyme in cog2159. The three-dimensional crystal structure of wild-type LigJ was determined in the presence [Protein Data Bank (PDB) entry 6DXQ ] and absence of the product CHA (PDB entry 6DWV ). The protein folds as a distorted (β/α)8-barrel, and a single zinc ion is bound in the active site at the C-terminal end of the central β-barrel. The product CHA is ligated to the zinc ion in the active site via the displacement of a single water molecule from the coordination shell of the metal center in LigJ. The product-bound structure reveals that the enzyme catalyzes the hydration of KCH with the formation of a chiral center at C4 with S stereochemistry. The E284Q mutant was unable to catalyze the hydration of KCH to CHA, and the structure of this mutant was determined in the presence of the substrate KCH (PDB entry 6DXS ). On the basis of the structure of LigJ in the presence of KCH and CHA, it is proposed that the side chain carboxylate of Glu-284 functions as a general base in the abstraction of a proton from a bound water molecule for nucleophilic attack at C4 of the substrate. The reaction is facilitated by the delocalization of the negative charge to the metal center via the carbonyl group at C2 of the substrate. C3 of the substrate is subsequently protonated by Glu-284 functioning as a general acid. The overall reaction occurs by the syn addition of water to the double bond between C4 and C3 of the substrate KCH. The kinetic constants for the hydration of KCH to CHA by LigJ at pH 8.0 are 25 s-1 ( kcat) and 2.6 × 106 M-1 s-1 ( kcat/ Km).

Anticalin® Proteins as Therapeutic Agents in Human Diseases.

Anticalin proteins are an emerging class of clinical-stage biopharmaceuticals with high potential as an alternative to antibodies. Anticalin molecules are generated by combinatorial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold dominated by a central β-barrel, supporting four structurally variable loops that form a binding site. Reshaping of this loop region results in Anticalin proteins that can recognize and tightly bind a wide range of medically relevant targets, from small molecules to peptides and proteins, as validated by X-ray structural analysis. Their robust format allows for modification in several ways, both as fusion proteins and by chemical conjugation, for example, to tune plasma half-life. Antagonistic Anticalin therapeutics have been developed for systemic administration (e.g., PRS-080: anti-hepcidin) or pulmonary delivery (e.g. PRS-060/AZD1402: anti-interleukin [IL]-4-Rα). Moreover, Anticalin proteins allow molecular formatting as bi- and even multispecific fusion proteins, especially in combination with antibodies that provide a second specificity. For example, PRS-343, which has recently entered clinical-stage development, combines an agonistic Anticalin targeting the costimulatory receptor 4-1BB with an antibody directed against the cancer antigen human epidermal growth factor receptor 2 (HER2), thus offering a novel treatment option in immuno-oncology.

Structural insight into a novel indole prenyltransferase in hapalindole-type alkaloid biosynthesis

FamD1 is a novel CloQ/NphB-family indole prenyltransferase which involves in hapalindole-type alkaloid biosynthesis. Here the native FamD1 structure and three protein-ligand complexes are analyzed to investigate the molecular basis of substrate binding and catalysis. FamD1 adopts a typical ABBA architecture of aromatic prenyltransferase, in which the substrate-binding chamber is found in the central β-barrel. The indole-containing acceptor substrate is bound adjacent to the prenyl donor. Based on the complex structures, a catalytic mechanism of FamD1 is proposed. Functional implications on the sister enzyme FamD2 are also discussed.

Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA

Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central β-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the β-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5′-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.

Self-Assembly and Conformational Changes of Hydrophobin Classes at the Air-Water Interface.

We use surface-specific vibrational sum-frequency generation spectroscopy (VSFG) to study the structure and self-assembling mechanism of the class I hydrophobin SC3 from Schizophyllum commune and the class II hydrophobin HFBI from Trichoderma reesei. We find that both hydrophobins readily accumulate at the water-air interface and form rigid, highly ordered protein films that give rise to prominent VSFG signals. We identify several resonances that are associated with β-sheet structures and assign them to the central β-barrel core present in both proteins. Differences between the hydrophobin classes are observed in their interfacial self-assembly. For HFBI, we observe no changes in conformation upon adsorption to the water surface. For SC3, we observe an increase in β-sheet-specific signals that supports a surface-driven self-assembly mechanism in which the central β-barrel remains intact and stacks into a larger-scale architecture, amyloid-like rodlets.

Biogenesis of beta-barrel proteins in evolutionary context

The vast majority of outer membrane (OM) proteins in Gram-negative bacteria belongs to the class of membrane-embedded β-barrel proteins. Besides Gram-negative bacteria, the presence of β-barrel proteins is restricted to the OM of the eukaryotic organelles mitochondria and chloroplasts that were derived from prokaryotic ancestors. The assembly of these proteins into the corresponding OM is in each case facilitated by a dedicated protein complex that contains a highly conserved central β-barrel protein termed BamA/YaeT/Omp85 in Gram-negative bacteria and Tob55/Sam50 in mitochondria. However, little is known about the exact mechanism by which these complexes mediate the integration of β-barrel precursors into the lipid bilayer. Interestingly, previous studies showed that during evolution, these complexes retained the ability to functionally assemble β-barrel proteins from different origins. In this review we summarize the current knowledge on the biogenesis pathway of β-barrel proteins in Gram-negative bacteria, mitochondria and chloroplasts and focus on the commonalities and divergences that evolved between the different β-barrel assembly machineries.

Solid-state NMR studies of metal-free SOD1 fibrillar structures

Copper-zinc superoxide dismutase 1 (SOD1) is present in the protein aggregates deposited in motor neurons of amyotrophic lateral sclerosis (ALS) patients. ALS is a neurodegenerative disease that can be either sporadic (ca. 90%) or familial (fALS). The most widely studied forms of fALS are caused by mutations in the sequence of SOD1. Ex mortuo SOD1 aggregates are usually found to be amorphous. In vitro SOD1, in its immature reduced and apo state, forms fibrillar aggregates. Previous literature data have suggested that a monomeric SOD1 construct, lacking loops IV and VII, (apoSODΔIV-VII), shares the same fibrillization properties of apoSOD1, both proteins having the common structural feature of the central β-barrel. In this work, we show that structural information can be obtained at a site-specific level from solid-state NMR. The residues that are sequentially assignable are found to be located at the putative nucleation site for fibrillar species formation in apoSOD, as detected by other experimental techniques.

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

High-resolution structure of a papaya plant-defence barwin-like protein solved by in-house sulfur-SAD phasing

The first crystal structure of a barwin-like protein, named carwin, has been determined at high resolution by single-wavelength anomalous diffraction (SAD) phasing using the six intrinsic S atoms present in the protein. The barwin-like protein was purified from Carica papaya latex and crystallized in the orthorhombic space group P212121. Using in-house Cu Kα X-ray radiation, 16 cumulative diffraction data sets were acquired to increase the signal-to-noise level and thereby the anomalous scattering signal. A sequence-database search on the papaya genome identified two carwin isoforms of 122 residues in length, both containing six S atoms that yield an estimated Bijvoet ratio of 0.93% at 1.54 Å wavelength. A systematic analysis of data quality and redundancy was performed to assess the capacity to locate the S atoms and to phase the data. It was observed that the crystal decay was low during data collection and that successful S-SAD phasing could be obtained with a relatively low data multiplicity of about 7. Using a synchrotron source, high-resolution data (1 Å) were collected from two different crystal forms of the papaya latex carwin. The refined structures showed a central β-barrel of six strands surrounded by several α-helices and loops. The β-barrel of carwin appears to be a common structural module that is shared within several other unrelated proteins. Finally, the possible biological function of the protein is discussed.

Norovirus RNA Synthesis Is Modulated by an Interaction between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1

Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027-13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse norovirus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the purified RdRps in vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR). Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.

Biogenesis of Mitochondria: Dual Role of Tom7 in Modulating Assembly of the Preprotein Translocase of the Outer Membrane

Biogenesis of the translocase of the outer mitochondrial membrane (TOM complex) involves the assembly of the central β-barrel forming protein Tom40 with six different subunits that are embedded in the membrane via α-helical transmembrane segments. The sorting and assembly machinery (SAM complex) of the outer membrane plays a central role in this process. The SAM complex mediates the membrane integration of β-barrel precursor proteins including Tom40. The small Tom proteins Tom5 and Tom6 associate with the precursor of Tom40 at the SAM complex at an early stage of the assembly process and play a stimulatory role in the formation of the mature TOM complex. A fraction of the SAM components interacts with the outer membrane protein mitochondrial distribution and morphology protein 10 (Mdm10) to form the SAM–Mdm10 machinery; however, different views exist on the function of the SAM–Mdm10 complex. We report here that the third small Tom protein, Tom7, plays an inhibitory role at two distinct steps in the biogenesis of the TOM complex. First, Tom7 plays an antagonistic role to Tom5 and Tom6 at the early stage of Tom40 assembly at the SAM complex. Second, Tom7 interacts with Mdm10 that is not bound to the SAM complex, and thus promotes dissociation of the SAM–Mdm10 complex. Since the SAM–Mdm10 complex is required for the biogenesis of Tom22, Tom7 delays the assembly of Tom22 with Tom40 at a late stage of assembly of the TOM complex. Thus, Tom7 modulates the biogenesis of topologically different proteins, the β-barrel forming protein Tom40 and Tom22 that contains a transmembrane α-helix.

Assembly of the Mitochondrial Protein Import Channel

The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central β-barrel channel, Tom40, and six proteins with α-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with α-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several α-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Δ mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.

The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase fromThermotoga maritimaas detected by NMR spectroscopy

The imidazole glycerol phosphate (ImGP) synthase from the hyperthermophilic bacterium Thermotoga maritima is a 1:1 complex of the glutaminase subunit HisH and the cyclase subunit HisF. It has been proposed that ammonia generated by HisH is transported through a channel to the active site of HisF, which generates intermediates of histidine (ImGP) and de novo biosynthesis of 5-aminoimidazole-4-carboxamideribotide. Solution NMR spectroscopy of ammonium chloride-titrated samples was used to study the interaction of NH(3) with amino acids inside this channel. Although numerous residues showed (15)N chemical shift changes, most of these changes were caused by nonspecific ionic strength effects. However, several interactions appeared to be specific. Remarkably, the amino acid residue Thr 78-which is located in the central channel-shows a large chemical shift change upon titration with ammonium chloride. This result and the reduced catalytic activity of the Thr78Met mutant indicate a special role of this residue in ammonia channeling. To detect and further characterize internal cavities in HisF, which might for example contribute to ammonia channeling, the interaction of HisF with the noble gas xenon was analyzed by solution NMR spectroscopy using (1)H-(15)N HSQC experiments. The results indicate that HisF contains three distinct internal cavities, which could be identified by xenon-induced chemical shift changes of the neighboring amino acid residues. Two of these cavities are located at the active site at opposite ends of the substrate N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binding groove. The third cavity is located in the interior of the central β-barrel of HisF and overlaps with the putative ammonia transport channel.

Crystal Structure of Lactadherin C2 Domain at 1.7Å Resolution with Mutational and Computational Analyses of Its Membrane-binding Motif

Lactadherin is a phosphatidyl-L-serine (Ptd-L-Ser)-binding protein that decorates membranes of milk fat globules. The major Ptd-l-Ser binding function of lactadherin has been localized to its C2 domain, which shares homology with the C2 domains of blood coagulation factor VIII and factor V. Correlating with this homology, purified lactadherin competes efficiently with factors VIII and V for Ptd-L-Ser binding sites, functioning as a potent anticoagulant. We have determined the crystal structure of the lactadherin C2 domain (Lact-C2) at 1.7A resolution. The bovine Lact-C2 structure has a beta-barrel core that is homologous with the factor VIII C2 (fVIII-C2) and factor V C2 (fV-C2) domains. Two loops at the end of the beta-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interactive residues of fVIII-C2 and fV-C2. In contrast to the corresponding loops in fVIII-C2 and fV-C2, spike 1 of Lact-C2 adopts a hairpin turn in which the 7-residue loop is stabilized by internal hydrogen bonds. Further, central glycine residues in two membrane-interactive loops may enhance conformability of Lact-C2 to membrane binding sites. Mutagenesis studies confirmed a membrane-interactive role for the hydrophobic and/or Gly residues of both spike 1 and spike 3. Substitution of spike 1 of fVIII-C2 into Lact-C2 also diminished binding. Computational ligand docking studies identified two prospective Ptd-l-Ser interaction sites. These results identify two membrane-interactive loops of Lact-C2 and provide a structural basis for the more efficient phospholipid binding of lactadherin as compared with factor VIII and factor V.

Protection of IgE-mediated allergic sensitization by active immunization with IgE loops constrained in GFP protein scaffold

Green fluorescent protein (GFP) exhibits a rigid central β-barrel, formed by eleven β-strands with floppy loops spanning between the stands. Herein, we evaluate whether the rigid β-barrel may serve as a scaffold that can constrain the loops of a foreign protein, and thus its antigenicity. The spanning loops, site 6 of GFP, were engineered with RE cloning sites for inserting oligonucleotides corresponding to FcɛRI-binding sequence of human IgE. In a high-throughput format, shortened oligonucleotides encoding eight amino acid residues of the receptor-binding regions were inserted into site 6 of GFP by PCR, followed by enabling sequences for in vitro transcription and translation at the 5′ end. Antigenized C2-3 linker (C2-3L) was shown by immuno-blots with polyclonal anti-IgE under native gel electrophoresis and transfer. Recombinant antigenized GFP was expressed and purified to homogeneity by metal affinity column, followed by Sephacryl S-200 high resolution gel filtration. Hyperimmune sera from mice immunized with C2-3L antigenized GFP contain anti-IgE reactive with JW8 murine/human chimeric IgE. Further, elevated serum anti-C2-3L and affinity pure antibodies effectively inhibits binding of JW8 IgE to recombinant FcɛRIα, and desensitizes JW8 to rat RBL-2H3 transfected with human FcɛRIα. This observation raised the possibility that active IgE vaccine may be employed in raising active protective anti-IgE in allergic patients as an alternative to passive immunization with MAb-E25 anti-IgE. Taken together, GFP appears suitable protein scaffold for spanning/constraining the C2-3L of human IgE as active vaccine; and this technique may be generally employed for eliciting antibodies to specific B-cell epitopes of other proteins.