Increased immunoaccessibility of MOMP epitopes in a vaccine formulated with amphipols may account for the very robust protection elicited against a vaginal challenge with Chlamydia muridarum.

There is a need to implement a vaccine to protect against Chlamydia trachomatis infections. To test a new vaccine, mice were immunized with the Chlamydia muridarum native major outer membrane protein (nMOMP) solubilized with either amphipol A8-35 or the detergent Z3-14. OVA was used as a negative control, and mice were inoculated intranasally with C. muridarum as positive controls. Animals vaccinated with nMOMP mounted strong Chlamydia-specific humoral and cell-mediated immune responses. Mice vaccinated with nMOMP/A8-35 had a higher ratio of Abs to denatured elementary bodies (EB) over live EB, recognized more synthetic MOMP peptides and had higher neutralizing titers than sera from mice immunized with nMOMP/Z3-14. T cell lymphoproliferative responses and levels of IFN-γ were also higher in mice vaccinated with nMOMP/A8-35 than with nMOMP/Z3-14. Following immunization, animals were challenged intravaginally with C. muridarum. On the basis of the number of mice with positive vaginal cultures, length of vaginal shedding, total number of positive vaginal cultures, and number of Chlamydia inclusion forming units recovered, nMOMP/A8-35 elicited a more robust protection than nMOMP/Z3-14. By depleting T cells with Abs, we determined that CD4(+) and not CD8(+) T cells mediated the protection elicited by nMOMP/A8-35. Mice were subsequently mated, and based on the number of pregnant mice and number of embryos, animals that were vaccinated with nMOMP/A8-35 or nMOMP/Z3-14 had fertility rates equivalent to the positive control group immunized with live EB and the fertility controls. In conclusion, increased accessibility of epitopes in the nMOMP/A8-35 preparation may account for the very robust protection against infection and disease elicited by this vaccine.

Glutamate provides a key structural contact between reticulon-4 (Nogo-66) and phosphocholine.

Human reticulon 4 (RTN-4) has been identified as the neurite outgrowth inhibitor (Nogo). This protein contains a span of 66 amino acids (Nogo-66) flanked by two membrane helices at the C-terminus. We previously determined the NMR structure of Nogo-66 in a native-like environment and defined the regions of Nogo-66 expected to be membrane embedded. We hypothesize that aromatic groups and a negative charge hyperconserved among RTNs (Glu26) drive the remarkably strong association of Nogo-66 with a phosphocholine surface. Glu26 is an isolated charge with no counterion provided by nearby protein groups. We modeled the docking of dodecylphosphocholine (DPC) with Nogo-66 and found that a lipid choline group could form a stable salt bridge with Glu26 and serve as a membrane anchor point. To test the role of the Glu26 anion in binding choline, we mutated this residue to alanine and assessed the structural consequences, the association with lipid and the affinity for the Nogo receptor. In an aqueous environment, Nogo-66 Glu26Ala is more helical than WT and binds the Nogo receptor with higher affinity. Thus, we can conclude that in the absence of a neutralizing positive charge provided by lipid, the glutamate anion is destabilizing to the Nogo-66 fold. Although the Nogo-66 Glu26Ala free energy of transfer from water into lipid is similar to that of WT, NMR data reveal a dramatic loss of tertiary structure for the mutant in DPC micelles. These data show that Glu26 has a key role in defining the structure of Nogo-66 on a phosphocholine surface. This article is part of a special issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Long-term stability of a vaccine formulated with the amphipol-trapped major outer membrane protein from Chlamydia trachomatis.

Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3-14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3-14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of (15)N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3-14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.

Dynamics and thermal stability of the bypass polymerase, DinB homolog (Dbh).

The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning over a wide range of temperatures. Existing X-ray structures were determined at temperatures where the protein is least active. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2°C-65°C) and metal ion binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbations with and without magnesium and manganese. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature. Hydrogen exchange protection factors and temperature coefficients reveal that both the thumb and finger domains are very dynamic relative to the palm and little-finger (LF) domains. These trends remain true at high temperature with dynamics increasing as temperatures increase from 35°C to 50°C. Notably, NMR spectra show that the Dbh tertiary structure cold denatures beginning at 25°C and increases in denaturation as the temperature is lowered to 5°C with little change observed by CD. Above 35°C, chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, without DNA bound. In contrast, these bound metals are not apparent in any Dbh crystal structures of the protein without DNA. Two ion binding sites are confirmed to be near the active site, as reported in other Y-family polymerases, and we report a novel ion binding site in the LF domain. Thus, the solution-state structure of the Dbh polymerase is distinct from that of the solid-state structures and shows an unusually high cold denaturation temperature.

Methemoglobin aggregation is modulated by the anti-sickling drug voxelotor.

Sickle cell disease is caused by a mutation in the beta subunit of hemoglobin (HbSS) that drives Hb fiber formation when the protein is in the deoxygenated (tense, T) state. The drug voxelotor was recently approved to treat sickle cell disease by preventing HbSS fiber formation. Voxelotor acts as an allosteric inhibitor of polymerization by maintaining the HbSS protein in the relaxed (R) conformation, limiting polymerization of T-state fibers. Normal blood cells contain small amounts of natural Hb fibers and a few percent of the Fe 3+ ferric form, metHb, incapable of binding oxygen. Although the drug Voxelotor is now in use, the effect of the drug on the oxidized metHb state has not been reported. Here we assessed the influence of voxelotor on normal human metHb. We compared the aggregation of metHb at two pH values (5.5 and 7.1). MetHb is known to form organized fiber structures at or below pH 5.5. We find that voxelotor significantly enhances fiber formation of metHb R-state at pH 5.5, consistent with the mode of action for this drug in maintaining the Hb R conformation. The opposite effect is observed at physiological pH values. Voxelotor significantly decreases the rate of metHb aggregate formation at pH 7.1 but did not affect protein stability. Notably, drug binding drives metHb into novel spherical particles with a morphology never seen before for Hb. The formation of these particles should be considered in patients being treated for sickle cell disease with voxelotor. WHY IT MATTERS: Voxelotor is an FDA-approved drug for sickle cell anemia, known to prevent hemoglobin fiber formation. Here, we investigate its effect on methemoglobin, the form of hemoglobin in which iron takes on the ferric Fe 3+ state. Our study examines voxelotor's impact on methemoglobin aggregation and stability. At pH 7.1, we found voxelotor to have an effect on methemoglobin solubility as evidenced by the formation of novel methemoglobin spherical structures. We observe that voxelotor significantly increases methemoglobin fiber formation at pH 5.5 but, notably, reduces methemoglobin aggregation at physiological pH levels. Minimal impact on methemoglobin thermodynamic stability is noted. These findings suggest voxelotor's potential therapeutic efficacy for various hemoglobinopathies, including conditions characterized by Heinz body formation.

Protein folding at the membrane interface, the structure of Nogo-66 requires interactions with a phosphocholine surface.

Repair of damage to the central nervous system (CNS) is inhibited by the presence of myelin proteins that prevent axonal regrowth. Consequently, growth inhibitors and their common receptor have been identified as targets in the treatment of injury to the CNS. Here we describe the structure of the extracellular domain of the neurite outgrowth inhibitor (Nogo) in a membrane-like environment. Isoforms of Nogo are expressed with a common C terminus containing two transmembrane (TM) helices. The ectodomain between the two TM helices, Nogo-66, is active in preventing axonal growth [GrandPre T, Nakamura F, Vartanian T, Strittmatter SM (2000) Nature 403:439-444]. We studied the structure of Nogo-66 alone and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles and dodecylphosphocholine (DPC) micelles as membrane mimetics. We find that Nogo-66 is largely disordered when free in solution. However, when bound to a phosphocholine surface Nogo-66 adopts a unique, stable fold, even in the absence of TM anchors. Using paramagnetic probes and protein-DPC nuclear Overhauser effects (NOEs), we define portions of the growth inhibitor likely to be accessible on the cell surface. With these data we predict that residues (28-58) are available to bind the Nogo receptor, which is entirely consistent with functional assays. Moreover, the conformations and relative positions of side chains recognized by the receptor are now defined and provide a foundation for antagonist design.

Multiple conformations of the cytidine repressor DNA-binding domain coalesce to one upon recognition of a specific DNA surface.

The cytidine repressor (CytR) is a member of the LacR family of bacterial repressors with distinct functional features. The Escherichia coli CytR regulon comprises nine operons whose palindromic operators vary in both sequence and, most significantly, spacing between the recognition half-sites. This suggests a strong likelihood that protein folding would be coupled to DNA binding as a mechanism to accommodate the variety of different operator architectures to which CytR is targeted. Such coupling is a common feature of sequence-specific DNA-binding proteins, including the LacR family repressors; however, there are no significant structural rearrangements upon DNA binding within the three-helix DNA-binding domains (DBDs) studied to date. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the CytR DBD free in solution and to determine the high-resolution structure of a CytR DBD monomer bound specifically to one DNA half-site of the uridine phosphorylase (udp) operator. We find that the free DBD populates multiple distinct conformations distinguished by up to four sets of NMR peaks per residue. This structural heterogeneity is previously unknown in the LacR family. These stable structures coalesce into a single, more stable udp-bound form that features a three-helix bundle containing a canonical helix-turn-helix motif. However, this structure differs from all other LacR family members whose structures are known with regard to the packing of the helices and consequently their relative orientations. Aspects of CytR activity are unique among repressors; we identify here structural properties that are also distinct and that might underlie the different functional properties.

Three arginine residues within the RGG box are crucial for ICP27 binding to herpes simplex virus 1 GC-rich sequences and for efficient viral RNA export.

ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A)(+) RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.

ICP27 phosphorylation site mutants display altered functional interactions with cellular export factors Aly/REF and TAP/NXF1 but are able to bind herpes simplex virus 1 RNA.

Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is phosphorylated. Phosphorylation can affect protein localization, protein interactions, and protein function. The major sites of ICP27 that are phosphorylated are serine residues 16 and 18, within a CK2 site adjacent to a leucine-rich region required for ICP27 export, and serine 114, within a PKA site in the nuclear localization signal. Viral mutants bearing serine-to-alanine or glutamic acid substitutions at these sites are defective in viral replication and gene expression. To determine which interactions of ICP27 are impaired, we analyzed the subcellular localization of ICP27 and its colocalization with cellular RNA export factors Aly/REF and TAP/NXF1. In cells infected with phosphorylation site mutants, ICP27 was confined to the nucleus even at very late times after infection. ICP27 did not colocalize with Aly/REF or TAP/NXF1, and overexpression of TAP/NXF1 did not promote the export of ICP27 to the cytoplasm. However, in vitro binding experiments showed that mutant ICP27 was able to bind to the same RNA substrates as the wild type. Nuclear magnetic resonance (NMR) analysis of the N terminus of ICP27 from amino acids 1 to 160, compared to mutants with triple substitutions to alanine or glutamic acid, showed that the mutations affected the overall conformation of the N terminus, such that mutant ICP27 was more flexible and unfolded. These results indicate that these changes in the structure of ICP27 altered in vivo protein interactions that occur in the N terminus but did not prevent RNA binding.

The HSV-1 ICP27 RGG box specifically binds flexible, GC-rich sequences but not G-quartet structures.

Herpes simplex virus 1 (HSV-1) protein ICP27, an important regulator for viral gene expression, directly recognizes and exports viral RNA through an N-terminal RGG box RNA binding motif, which is necessary and sufficient for RNA binding. An ICP27 N-terminal peptide, including the RGG box RNA binding motif, was expressed and its binding specificity was analyzed using EMSA and SELEX. DNA oligonucleotides corresponding to HSV-1 glycoprotein C (gC) mRNA, identified in a yeast three-hybrid analysis, were screened for binding to the ICP27 N-terminal peptide in EMSA experiments. The ICP27 N-terminus was able to bind most gC substrates. Notably, the ICP27 RGG box was unable to bind G-quartet structures recognized by the RGG domains of other proteins. SELEX analysis identified GC-rich RNA sequences as a common feature of recognition. NMR analysis of SELEX and gC sequences revealed that sequences able to bind to ICP27 did not form secondary structures and conversely, sequences that were not able to bind to ICP27 gave spectra consistent with base-pairing. Therefore, the ICP27 RGG box is unique in its recognition of nucleic acid sequences compared to other RGG box proteins; it prefers flexible, GC-rich substrates that do not form stable secondary structures.

Markov state models and NMR uncover an overlooked allosteric loop in p53.

The tumor suppressor p53 is the most frequently mutated gene in human cancer, and thus reactivation of mutated p53 is a promising avenue for cancer therapy. Analysis of wildtype p53 and the Y220C cancer mutant long-timescale molecular dynamics simulations with Markov state models and validation by NMR relaxation studies has uncovered the involvement of loop L6 in the slowest motions of the protein. Due to its distant location from the DNA-binding surface, the conformational dynamics of this loop has so far remained largely unexplored. We observe mutation-induced stabilization of alternate L6 conformations, distinct from all experimentally-determined structures, in which the loop is both extended and located further away from the DNA-interacting surface. Additionally, the effect of the L6-adjacent Y220C mutation on the conformational landscape of the functionally-important loop L1 suggests an allosteric role to this dynamic loop and the inactivation mechanism of the mutation. Finally, the simulations reveal a novel Y220C cryptic pocket that can be targeted for p53 rescue efforts. Our approach exemplifies the power of the MSM methodology for uncovering intrinsic dynamic and kinetic differences among distinct protein ensembles, such as for the investigation of mutation effects on protein function.

Improved protection against Chlamydia muridarum using the native major outer membrane protein trapped in Resiquimod-carrying amphipols and effects in protection with addition of a Th1 (CpG-1826) and a Th2 (Montanide ISA 720) adjuvant.

A new vaccine formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) and amphipols was assessed in an intranasal (i.n.) challenge mouse model. nMOMP was trapped either in amphipol A8-35 (nMOMP/A8-35) or in A8-35 conjugated with Resiquimod (nMOMP/Resiq-A8-35), a TLR7/8 agonist added as adjuvant. The effects of free Resiquimod and/or additional adjuvants, Montanide ISA 720 (TLR independent) and CpG-1826 (TLR9 agonist), were also evaluated. Immunization with nMOMP/A8-35 alone administered i.n. was used as negative adjuvant-control group, whereas immunizations with C. muridarum elementary bodies (EBs) and MEM buffer, administered i.n., were used as positive and negative controls, respectively. Vaccinated mice were challenged i.n. with C. muridarum and changes in body weight, lungs weight and recovery of Chlamydia from the lungs were evaluated. All the experimental groups showed protection when compared with the negative control group. Resiquimod alone produced weak humoral and cellular immune responses, but both Montanide and CpG-1826 showed significant increases in both responses. The addition of CpG-1826 alone switched immune responses to be Th1-biased. The most robust protection was elicited in mice immunized with the three adjuvants and conjugated Resiquimod. Increased protection induced by the Resiquimod covalently linked to A8-35, in the presence of Montanide and CpG-1826 was established based on a set of parameters: (1) the ability of the antibodies to neutralize C. muridarum; (2) the increased proliferation of T-cells in vitro accompanied by higher production of IFN-γ, IL-6 and IL-17; (3) the decreased body weight loss over the 10 days after challenge; and (4) the number of IFUs recovered from the lungs at day 10 post challenge. In conclusion, a vaccine formulated with the C. muridarum nMOMP bound to amphipols conjugated with Resiquimod enhances protective immune responses that can be further improved by the addition of Montanide and CpG-1826.

pH dependence of sphingosine aggregation.

Sphingosine and sphingosine 1-phosphate (S1P) are sphingolipid metabolites that act as signaling messengers to activate or inhibit multiple downstream targets to regulate cell growth, differentiation, and apoptosis. The amphiphilic nature of these compounds leads to aggregation above their critical micelle concentrations (CMCs), which may be important for understanding lysosomal glycosphingolipid storage disorders. We investigated the aggregation of sphingosine and S1P over a comprehensive, physiologically relevant range of pH values, ionic strengths, and lipid concentrations by means of dynamic light scattering, titration, and NMR spectroscopy. The results resolve discrepancies in literature reports of CMC and pK(a) values. At physiological pH, the nominal CMCs of sphingosine and S1P are 0.99 +/- 0.12 microM (pH 7.4) and 14.35 +/- 0.08 microM (pH 7.2), respectively. We find that pH strongly affects the aggregation behavior of sphingosine by changing the ionic and hydrogen-bonding states; the nominal critical aggregation concentrations of protonated and deprotonated sphingosine are 1.71 +/- 0.24 microM and 0.70 +/- 0.02 microM, respectively. NMR measurements revealed that the NH3+-NH2 transition of sphingosine occurs at pH 6.6, and that there is a structural shift in sphingosine aggregates caused by a transition in the predominant hydrogen-bonding network from intramolecular to intermolecular that occurs between pH 6.7 and 9.9.

Chemical shift mapping of gammadelta resolvase dimer and activated tetramer: mechanistic implications for DNA strand exchange.

Several static structural models exist for gammadelta resolvase, a self-coded DNA recombinase of the gammadelta transposon. While these reports are invaluable to formulation of a mechanistic hypothesis for DNA strand exchange, several questions remain. Foremost among them concerns the protomer structural dynamics within the protein/DNA synaptosome. Solution NMR chemical shift assignments have been made for truncated variants of the natural wild-type dimer, which is inactive without the full synaptosome structure, and a mutationally activated tetramer. Of the 134 residues, backbone (1)H, (15)N, and (13)Calpha assignments are made for 121-124 residues in the dimer, but only 76-80 residues of the tetramer. These assignment differences are interpreted by comparison to X-ray diffraction models of the recombinase dimer and tetramer. Inspection of intramolecular and intermolecular structural variation between these models suggests a correspondence between sequence regions at subunit interfaces unique to tetramer, and the regions that can be sequentially assigned in the dimer but not the tetramer. The loss of sequential context for assignment is suggestive of stochastic fluctuation between structural states involving protomer-protomer interactions exclusive to the activated tetrameric state, and may be indicative of dynamics which pertain to the recombinase mechanism.

Electropositive charge in alpha-defensin bactericidal activity: functional effects of Lys-for-Arg substitutions vary with the peptide primary structure.

Cationic amino acids contribute to alpha-defensin bactericidal activity. Curiously, although Arg and Lys have equivalent electropositive charges at neutral pH, alpha-defensins contain an average of nine Arg residues per Lys residue. To investigate the role of high alpha-defensin Arg content, all Arg residues in mouse Paneth cell alpha-defensin cryptdin 4 (Crp4) and rhesus myeloid alpha-defensin 4 (RMAD-4) were replaced with Lys to prepare (R/K)-Crp4 and (R/K)-RMAD-4, respectively. Lys-for-Arg replacements in Crp4 attenuated bactericidal activity and slowed the kinetics of Escherichia coli ML35 cell permeabilization, and (R/K)-Crp4 required longer exposure times to reduce E. coli cell survival. In marked contrast, Lys substitutions in RMAD-4 improved microbicidal activity against certain bacteria and permeabilized E. coli more effectively. Therefore, Arg-->Lys substitutions attenuated activity in Crp4 but not in RMAD-4, and the functional consequences of Arg-->Lys replacements in alpha-defensins are dependent on the peptide primary structure. In addition, the bactericidal effects of (R/K)-Crp4 and (R/K)-RMAD-4 were more sensitive to inhibition by NaCl than those of the native peptides, suggesting that the high Arg content of alpha-defensins may be under selection to confer superior microbicidal function under physiologic conditions.

Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation.

The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.

Co-delivery of amphipol-conjugated adjuvant with antigen, and adjuvant combinations, enhance immune protection elicited by a membrane protein-based vaccine against a mucosal challenge with Chlamydia.

INTRODUCTION: Chlamydial infections are spread worldwide and a vaccine is needed to control this pathogen. The goals of this study were to determine if the delivery of an adjuvant associated to the antigen, via a derivatized amphipol, and adjuvant combinations improve vaccine protection. METHODS: A novel approach, trapping the Chlamydia muridarum (Cm) native MOMP (nMOMP) with amphipols (A8-35), bearing a covalently conjugated peptide (EP67), was used. Adjuvants incorporated were: EP67 either conjugated to A8-35, which was used to trap nMOMP (nMOMP/EP67-A8-35), or free as a control, added to nMOMP/A8-35 complexes (nMOMP/A8-35+EP67); Montanide ISA 720 to enhance humoral responses, and CpG-1826 to elicit robust cell-mediated immunity (CMI). BALB/c mice were immunized by mucosal and systemic routes. Intranasal immunization with live Cm was used as positive control and three negative controls were included. Mice were challenged intranasally with Cm and changes in body weight, lungs weight and number of Cm-inclusion forming units (IFU) recovered from the lungs were evaluated to establish protection. To assess local responses levels of IFN- γ and Cm-specific IgA were determined in lungs' supernatants. RESULTS: Structural assays demonstrated that nMOMP secondary structure and thermal stability were maintained when A8-35 was covalently modified. Mice vaccinated with nMOMP/EP67-A8-35 were better protected than animals immunized with nMOMP/A8-35+EP67. Addition of Montanide enhanced Th2 responses and improved protection. Including CpG-1826 further broadened, intensified and switched to Th1-biased immune responses. With delivery of nMOMP and the three adjuvants, as determined by changes in body weight, lungs weight and number of IFU recovered from lungs, protection at 10 days post-challenge was equivalent to that induced by immunization with live Cm. CONCLUSIONS: Covalent association of EP67 to A8-35, used to keep nMOMP water-soluble, improves protection over that conferred by free EP67. Adjuvant combinations including EP67+Montanide+CpG-1826, by broadening and intensifying cellular and humoral immune responses, further enhanced protection.

Flexibility and adaptability in binding of E. coli cytidine repressor to different operators suggests a role in differential gene regulation.

Interactions between DNA-bound transcription factors CytR and CRP regulate the promoters of the Escherichia coli CytR regulon. A distinctive feature of the palindromic CytR operators is highly variable length central spacers (0-9 bp). Previously we demonstrated distinct modes of CytR binding to operators that differ in spacer length. These different modes are characterized by opposite enthalpic and entropic contributions at 25 degrees C. Of particular note were radically different negative DeltaCp values suggesting variable contribution from coupled protein folding and/or DNA structural transitions. We proposed that the CytR DNA binding-domain adopts either a more rigid or flexible DNA-bound conformation in response to the different spacer lengths. More recently, similar effects were shown to contribute to discrimination between operator and non-specific DNA binding by LacR, a CytR homolog. Here we have extended the thermodynamic analysis to the remaining natural CytR operators plus a set of synthetic operators designed to isolate spacing as the single variable. The thermodynamic results show a broad and monotonic range of effects that are primarily dependent on spacer length. The magnitude of effects suggests participation by more than the DNA-binding domain. 15N HSQC NMR and CD spectral analyses were employed to characterize the structural basis for these effects. The results indicate that while CytR forms a well-ordered structure in solution, it is highly dynamic. We propose a model in which a large ensemble of native state conformations narrows upon binding, to an extent governed by operator spacing. This in turn is expected to constrain intermolecular interactions in the CytR-CRP-DNA complex, thus generating operator-specific effects on repression and induction of transcription.

Exploring the interaction between the protein kinase A catalytic subunit and caveolin-1 scaffolding domain with shotgun scanning, oligomer complementation, NMR, and docking.

The techniques of phage-displayed homolog shotgun scanning, oligomer complementation, NMR secondary structure analysis, and computational docking provide a complementary suite of tools for dissecting protein-protein interactions. Focusing these tools on the interaction between the catalytic sub-unit of protein kinase A (PKAcat) and caveolin-1 scaffolding domain (CSD) reveals the first structural model for the interaction. Homolog shotgun scanning varied each CSD residue as either a wild-type or a homologous amino acid. Wild-type to homolog ratios from 116 different homologous CSD variants identified side-chain functional groups responsible for precise contacts with PKAcat. Structural analysis by NMR assigned an alpha-helical conformation to the central residues 84- 97 of CSD. The extensive mutagenesis data and NMR secondary structure information provided constraints for developing a model for the PKAcat-CSD interaction. Addition of synthetic CSD to phage-displayed CSD resulted in oligomer complementation, or enhanced binding to PKAcat. Together with previous experiments examining the interaction between CSD and endothelial nitric oxide synthase (eNOS), the results suggest a general oligomerization-dependent enhancement of binding between signal transducing enzymes and caveolin-1.

Specific interactions of distamycin with G-quadruplex DNA.

Distamycin binds the minor groove of duplex DNA at AT-rich regions and has been a valuable probe of protein interactions with double-stranded DNA. We find that distamycin can also inhibit protein interactions with G-quadruplex (G4) DNA, a stable four-stranded structure in which the repeating unit is a G-quartet. Using NMR, we show that distamycin binds specifically to G4 DNA, stacking on the terminal G-quartets and contacting the flanking bases. These results demonstrate the utility of distamycin as a probe of G4 DNA-protein interactions and show that there are (at least) two distinct modes of protein-G4 DNA recognition which can be distinguished by sensitivity to distamycin.