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To assure the safety of oxide-fuel based nuclear reactors, the knowledge of the atomic-scale properties of U1−yMyO2±x materials is essential. These compounds show complex chemical properties, originating from the fact that actinides and rare earths may occur with different oxidation states. In these mostly ionic materials, aliovalent cationic configurations can induce changes in the oxygen stoichiometry, with dramatic effects on the properties of the fuel. First studies on U1−yAmyO2±x indicated that these materials exhibit particularly complex electronic and local-structure configurations. Here we present an in-depth study of these compounds, over a wide compositional domain, by combining XRD, XAS and Raman spectroscopy. We provide evidences of the co-existence of four different cations (U4+, U5+, Am3+, Am4+) in U1−yMyO2±x compounds, which nevertheless maintain the fluorite structure. Indeed, we show that the cationic sublattice is basically unaffected by the extreme multi-valence states, whereas complex defects are present in the oxygen sublattice. Metal oxidation state in complex actinide oxides may have drastic effects on material properties. Here, the authors use a range of techniques to analyse U1−yAmyO2±x and find that while multiple cations are present the fluorite structure is maintained albeit with complex defects in the oxygen sublattice. [ABSTRACT FROM AUTHOR]

The present study consisted of the development and validation of a novel multi-rater Domain-Specific School Diversity Model scale (DS-SDM) that captures pupils' and teachers' perceptions of the extent to which their school adopts assimilationist, colorblind, and pluralist approaches to the domains of (a) languages, (b) religions, (c) the curriculum, and (d) students' identities. Using data collected from 3073 students ages 10-12 years and 816 teachers from 59 primary schools in Flanders, Belgium, we performed multilevel exploratory factor analyses and confirmatory factor analyses to evaluate these novel scales. We identified a total of nine measurement scales among students and seven among teachers that were reliable (internal consistency range = 0.63-0.86) and invariant across different subgroups of pupils (i.e., ethnic minoritized versus majority pupils). Interestingly, we found that perceptions by pupils and teachers within the same school were often notably different, especially in the domains of curriculum and identities. Overall, this study offers novel tools to capture perceptions of school diversity models among students and teachers in more nuanced and domain-specific ways. As such, it paves the way for future school psychological or educational studies and interventions targeting domain-specific diversity practices that are most strongly related to ethnic minoritized and majority students' well-being and school achievement., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Society for the Study of School Psychology. Published by Elsevier Ltd. All rights reserved.)

The thermodynamic and thermo-physical properties of the binary salt systems AECl 2 -NdCl 3 (AE = Sr, Ba) have been investigated using an experimental and modelling approach. The binary salt systems both include a single intermediate salt, i.e. Sr 9 Nd 5 Cl 33 and Ba 3 Nd 2 Cl 12 , respectively. The structure of these intermediates has been investigated with X-ray diffraction (XRD). Furthermore, these systems exhibit mutual solubility of NdCl 3 in BaCl 2 and SrCl 2 . The investigation of these solid solutions has been performed using quenching experiments and subsequent post-characterisation by XRD. Phase diagram equilibria have also been investigated using differential scanning calorimetry (DSC). Using the aforementioned information on phase transitions, intermediate compound formation, and solid solubility, thermodynamic assessment of the systems has been performed using the CALPHAD method. The model for the Gibbs energy of the liquid solution is the quasi-chemical formalism in the quadruplet approximation, while the model for the Gibbs energy of the solid solutions is the two-sublattice polynomial model.

The transmutation of minor actinides (in particular, Np and Am), which are among the main contributors to spent fuel α-radiotoxicity, was studied in the SUPERFACT irradiation. Several types of transmutation UO 2 -based fuels were produced, differing by their minor actinide content ( 241 Am, 237 Np, Pu), and irradiated in the Phénix fast reactor. Due to the high content in rather short-lived alpha-decaying actinides, both the archive, but also the irradiated fuels, cumulated an alpha dose during a laboratory time scale, which is comparable to that of standard LWR fuels during centuries/millenaries of storage. Transmission Electron Microscopy was performed to assess the evolution of the microstructure of the SUPERFACT archive and irradiated fuel. This was compared to conventional irradiated spent fuel (i.e., after years of storage) and to other 238 Pu-doped UO 2 for which the equivalent storage time would span over centuries. It could be shown that the microstructure of these fluorites does not degrade significantly from low to very high alpha-damage doses, and that helium bubbles precipitate.

The short-range structures of LiF-ThF 4 , NaF-AnF 4 , KF-AnF 4 , and Cs-AnF 4 (An = Th, U), were probed using in situ high temperature Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy. Signally, the EXAFS spectra of pure molten ThF 4 and UF 4 were measured for the first time. The data were interpreted with the aid of Molecular Dynamics (MD) and standard fitting of the EXAFS equation. As in related studies, a speciation distribution dominated by [AnF x ] 4-x (x = 7, 8, 9) coordination complexes was observed. The average coordination number was found to decrease with the increasing size of the alkali cation, and increase with AnF 4 content. An average coordination number close to 6, which had not been detected before in melts of alkali actinide fluorides, was seen when CsF was used as solvent.

Cesium and iodine, which are formed during a fission process in a nuclear reactor, are considered as major fission products responsible for the environmental burden in case of a nuclear accident. From the safety point of view, it is thus important to understand their release mechanism when overheating of the reactor core occurs. This work presents an experimental investigation of the behaviour of caesium iodide and caesium fluoride in fluoride based molten salt reactor fuel during high temperature events. It has been demonstrated that CsF will be retained in the fuel salt and thus its volatility will be significantly reduced, while CsI will not dissolve in the fluoride-based fuel matrix and will thus remain more volatile. The influence of the presence of CsI and CsF on the melting behaviour of the fuel has been investigated using calorimetry, revealing their negligible effects.

Background: During their specialty training program residents are stimulated to think and work in a goal-oriented way. Patient 'no shows' are quite common in the mental healthcare system, consequently causing the ineffective use of healthcare services.
AIM: To reduce the amount of 'no shows' in an outpatient clinic for hospital psychiatry by sending reminders via text messaging.
METHOD: A quasi-experimental study was conducted at an outpatient clinic for hospital-psychiatry in 2016, in which 101 patients were included. Eventually, 50 patients received a text message to remind them of their appointment, while 46 did not. We used a χ2 test to evaluate group differences. The effect size was expressed in the 'number needed to cash' (nnc), similar to the number needed to treat (nnt). Routinely available hospital-data was used to estimate lost revenue per year.
RESULTS: A significant group difference was found in the number of outpatient clinic visits in favour of sending a text message reminder (74% vs. 92%, p = 0.018). This corresponded to a nnc of 5.53, i.e. 6 text messages need to be sent in order to accomplish one extra patient showing up for their intake. Based on hospital-data from 2016 the estimated lost revenue was € 53.017,38 / year at our outpatient clinic.
CONCLUSION: Sending reminders via text messaging is effective in reducing the number of 'no shows' at an outpatient clinic for hospital psychiatry.

Major and severe accidents have occurred three times in nuclear power plants (NPPs), at Three Mile Island (USA, 1979), Chernobyl (former USSR, 1986) and Fukushima (Japan, 2011). Research on the causes, dynamics, and consequences of these mishaps has been performed in a few laboratories worldwide in the last three decades. Common goals of such research activities are: the prevention of these kinds of accidents, both in existing and potential new nuclear power plants; the minimization of their eventual consequences; and ultimately, a full understanding of the real risks connected with NPPs. At the European Commission Joint Research Centre's Institute for Transuranium Elements, a laser-heating and fast radiance spectro-pyrometry facility is used for the laboratory simulation, on a small scale, of NPP core meltdown, the most common type of severe accident (SA) that can occur in a nuclear reactor as a consequence of a failure of the cooling system. This simulation tool permits fast and effective high-temperature measurements on real nuclear materials, such as plutonium and minor actinide-containing fission fuel samples. In this respect, and in its capability to produce large amount of data concerning materials under extreme conditions, the current experimental approach is certainly unique. For current and future concepts of NPP, example results are presented on the melting behavior of some different types of nuclear fuels: uranium-plutonium oxides, carbides, and nitrides. Results on the high-temperature interaction of oxide fuels with containment materials are also briefly shown.

A new double neptunium zirconium phosphate of the type M x Zr 2 (PO 4 ) 3 (M = Np), crystallizing in the structure type NaZr 2 (PO 4 ) 3 (NZP, NASICON), was synthesized by solid state reactions at high temperatures and characterized by X-ray diffraction, infrared spectroscopy and Mössbauer spectroscopy. The Rietveld refinement of the XRD pattern together with the analysis of the IR spectra of the sample confirmed the space group P3[combining macron]c, the same as that for the lanthanide analogues Ln 0.33 Zr 2 (PO 4 ) 3 . However, Mössbauer studies revealed the presence of neptunium in the two oxidation states +3 and +4, indicating a two-phase NZP system with different crystallographic environments of the neptunium atoms. The thermal behaviour of the sample was followed up to 1400 °C by thermogravimetric analysis.

Introduction: In this study we evaluated the value of pre-operative glucose corrected maximum standard uptake value (GC-SUVmax) as prognostic factor in patients with early stage non-small cell lung cancer (NSCLC) after complete surgical resection., Methods: This study was designed as a retrospectively evaluated single center study with prospective data registry. Inclusion criteria were: histologically proven stage I NSCLC, 18F-FDG-PET/CT scan prior to surgery, complete resection (R0) and follow up in our outpatient department. Exclusion criteria were: history of malignancy other than NSCLC, diabetes and (neo) adjuvant therapy. Follow up period was 5 years., Results: Between 2006 and 2008 a total of 33 patients (16 males, 17 females) met the inclusion criteria. SUVmax and GC-SUVmax were strongly correlated (Spearman's ρ = 0.97). Five-year overall survival (OS) rate was 70 % (95 % CI = 56-87 %). Patients who died within 5 years of follow up had significantly higher pre-operative GC-SUVmax (median = 10.6, IQR = 8.3-14.4) than patients who were alive at 5-year follow up (median = 6.4, IQR = 3.0-9.8), p = 0.04. SUVmax showed similar differences: 10.4 (8-12.9) vs. 6.6 (3.0-8.8), p = 0.047. The area under the receiver-operating characteristic (ROC) curve at 5 years was 0.70 (95 % CI = 0.50-0.90) for GC-SUVmax and 0.71 (95 % CI = 0.51-0.91) for SUVmax (p = 0.75)., Conclusion: Pre-operative FDG tumor uptake in patients with NSCLC is predictive for survival after complete surgical resection. GC-SUVmax, as an additional value to SUVmax, may better approach competitive inhibition of FDG and glucose in tumors, however, in this study this potential advantage, if any, was very small.

The applicability and limitations of sulphur isotope ratio as a nuclear forensic signature have been studied. The typically applied leaching methods in uranium mining processes were simulated for five uranium ore samples and the n ( 34 S)/ n ( 32 S) ratios were measured. The sulphur isotope ratio variation during uranium ore concentrate (UOC) production was also followed using two real-life sample sets obtained from industrial UOC production facilities. Once the major source of sulphur is revealed, its appropriate application for origin assessment can be established. Our results confirm the previous assumption that process reagents have a significant effect on the n ( 34 S)/ n ( 32 S) ratio, thus the sulphur isotope ratio is in most cases a process-related signature.

Background: We investigated the prevalence and protective value of serosorting [ie, establishing HIV concordance in advance to practice unprotected anal intercourse (UAI)] with casual partners (CP) among HIV-negative men who have sex with men (MSM) using longitudinal data from 2007 to 2011., Methods: Men of the Amsterdam Cohort Studies were tested biannually for HIV-1 antibodies and filled in questionnaires about sexual behavior in the preceding 6 months. HIV incidence was examined among men who practiced UAI, UAI with serosorting, or consistent condom use, using Poisson regression., Results: Of 445 MSM with CPs, 31 seroconverted for HIV during a total follow-up of 1107 person-years. Overall observed HIV incidence rate was 2.8/100 person-years. Consistent condom use was reported in 64%, UAI in 25%, and UAI with serosorting in 11% of the 2137 follow-up visits. MSM who practiced serosorting were less likely to seroconvert [adjusted incidence rate ratio (aIRR) = 0.46; 95% confidence interval (CI): 0.13 to 1.59] than MSM who had UAI, but more likely to seroconvert than MSM who consistently used condoms (aIRR = 1.32; 95% CI: 0.37 to 4.62), although differences in both directions were not statistically significant. MSM who consistently used condoms were less likely to seroconvert than MSM who had UAI (aIRR = 0.37; 95% CI: 0.18 to 0.77)., Discussion: The protective effect for serosorting we found was not statistically significant. Consistent condom use was found to be most protective against HIV infection. Larger studies are needed to demonstrate whether serosorting with CPs offers sufficient protection against HIV infection, and if not, why it fails to do so.

Purpose. To describe the treatment of renal artery thrombosis with ultrasound-accelerated thrombolysis and discuss the management of prolonged renal ischemia. Case. A 76-year-old patient with a single functional kidney, mild chronic renal impairment, and a recent history of endovascular repair of a thoracoabdominal aneurysm with an aortic branch graft presented with acute flank pain, anuria, and renal failure. The side branch from the aortic stent graft to his single, right, functional kidney appeared to be completely thrombosed. Symptoms had started after cessation of oral anticoagulants because of a planned mastectomy for breast cancer. After identification of the occlusion, ultrasound-accelerated thrombolysis was started 19 hours after the onset of anuria. Angiography, 4 hours after beginning of therapy, already showed partial dissolution of the thrombus and angiographic control after 18 hours showed complete patency of the renal artery side branch. Despite a long period of ischemia, renal function was completely recovered. Conclusion. In patients with acute renal ischemia due to thrombosis of the renal artery, complete recovery of function can be achieved with ultrasound-accelerated thrombolysis, even after prolonged periods of ischemia.

Purpose: To describe a novel hybrid technique to address two challenges in endovascular repair of chronic dissecting thoracic aortic aneurysm (dTAA): obtaining an adequate seal of the stent-graft in a half-moon-shaped fibrotic aortic lumen and preserving flow into the distal true and false lumens., Technique: The technique is demonstrated in a 52-year-old man who presented with progressive asymptomatic dilatation of the thoracic aorta 9 years after undergoing a Bentall procedure for a Stanford type A dissection followed by arch replacement and elephant trunk construction. Imaging at this admission showed a 6.8-cm dissecting aneurysm extending distally to ∼4 cm above the celiac trunk; the dissection included both common iliac arteries. The patient refused a thoracotomy, so a hybrid procedure was devised to resect the intimal flap via a median subxyphoid incision and transperitoneal approach through the lesser sac. Two overlapping Zenith TX-2 stent-grafts were deployed into the elephant trunk, terminating just above the surgically created "flow divider" at the level of the celiac trunk. Imaging showed adequate sealing at both ends of the stent-graft and a type II endoleak that persisted into follow-up, but the aneurysm diameter decreased to 6.4 cm, and there was unobstructed flow into the visceral, renal, and iliac arteries., Conclusion: In this case of chronic dTAA, open surgical removal of a segment of the dissection flap via a subxyphoid incision provided a distal landing zone for subsequent endoluminal repair, with exclusion of the aneurysm and preservation of antegrade flow in both true and false lumens.

Pfs48/45 is an important transmission-blocking vaccine candidate antigen of the human malaria parasite Plasmodium falciparum. This study was aimed at synthesis of recombinant Pfs48/45 containing conformation-constrained epitopes of the native antigen in yeast. Since in the yeast Saccharomyces cerevisiae induction of gene-expression led to prematurely terminated transcripts, an entirely synthetic gene of higher GC content was assembled. Replacement of the AT rich natural gene by the synthetic gene relieved the observed premature transcription termination. Nevertheless, recombinant protein expression could not be detected. In contrast, in the yeast Pichia pastoris low levels of recombinant Pfs48/45 were produced upon induction of synthetic gene expression. The recombinant protein was shown to be disulphide-bridge constrained, but was not recognised by transmission-blocking antibodies and did not induce transmission-blocking sera in mice.

Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of the pfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activates pfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum.

With the aim of developing transmission-blocking vaccines based on the sexual stage-specific surface antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum, the gene encoding Pfs48/45 was incorporated into the genome of a recombinant vaccinia virus. In virus-infected mammalian tissue culture cells, recombinant Pfs48/45 antigen (rPfs48/45) is posttranslational modified to produce a highly N-glycosylated polypeptide. The rPfs48/45 protein was radiolabeled with ethanolamine, consisting of a further posttranslational modification in the form of a glycosylphosphatidylinositol anchor at its carboxy-terminal end. The rPfs48/45 was not detected on the surface of the infected cells; instead, it remained within the secretion pathway of mammalian cells irrespective of the duration of infection or culture temperature. Studies with monoclonal antibodies specific for disulfide band-dependent epitopes of Pfs48/45 revealed that recombinant Pfs48/45 is not folded in its authentic conformation even if N-glycosylation was chemically inhibited. Infection of mice and rabbits with recombinant virus elicited Pfs48/45-specific antibodies; however, the antisera failed to block parasite transmission in a standard mosquito membrane-feeding assay., (Copyright 1998 Academic Press.)

The three-dimensional structure of the major coat protein of bacteriophage M13, solubilized in detergent micelles, has been determined using heteronuclear multidimensional NMR and restrained molecular dynamics. The protein consists of two alpha-helices, running from residues 8 to 16 and 25 to 45, respectively. These two helices are connected by a flexible and distorted helical hinge region. The structural properties of the coat protein make it resemble a flail, in which the hydrophobic helix (residues 25 to 45) is the handle and the other, amphipathic, helix the swingle. In this metaphor, the hinge region is the connecting piece of leather. The mobility of the residues in the hinge region is likely to enable a smooth transformation from the membrane-bound form, mimicked by the structure in detergent micelles, into the structure in the mature phage. A specific distribution of the residues over the surface of the two helices was observed in the presented high-resolution structure of the membrane-bound form of the major coat protein as well as in the structure in the mature phage. All data suggest that this arrangement of residues is important for the interactions of the protein with the membrane, for correct protein-DNA and protein-protein interactions in the phage and for a proper growth of the phage during the assembly process. By combining our findings with earlier NMR results on the major coat protein in detergent micelles, we were able to construct a model that addresses the role of specific residues in the assembly process., (Copyright 1998 Academic Press.)

A precondition for the development of a transmission blocking vaccine based on the sexual stage-specific surface antigen Pfs48/45 of Plasmodium falciparum is its heterologous synthesis in a native state. Here we describe the production of recombinant Pfs48/45 in Escherichia coli. Two recombinant proteins, of which one is a glutathione-S-transferase fusion protein, were produced. Enzyme-linked immunosorbent assays showed that at least a subfraction of the recombinant proteins had a conformation capable of binding transmission blocking monoclonal antibodies. However, despite the fact that both proteins were very immunogenic, they did not induce transmission blocking immunity in mice or rabbits. Immunological studies with congenic mouse strains demonstrated that immune responses could be boosted with gametocyte extracts and were not restricted to a particular class II major histocompatibility complex haplotype.

The solution structure of the 18-kDa single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been refined using 40 ms 15N- and 13C-edited NOESY spectra and many homo- and heteronuclear J-couplings. The structures are highly precise, but some variation was found in the orientation of the beta-hairpin denoted the DNA binding wing with respect to the core of the protein. Backbone dynamics of the protein was investigated in the presence and absence of DNA by measuring the R1 and R2 relaxation rates of the 15N nuclei and the 15N-1H NOE. It was found that the DNA binding wing is much more flexible than the rest of the protein, but its mobility is largely arrested upon binding of the protein to d(A)6. This confirms earlier hypotheses on the role of this hairpin in the function of the protein, as will be discussed. Furthermore, the complete DNA binding domain of the protein has been mapped by recording two-dimensional TOCSY spectra of the protein in the presence and absence of a small amount of spin-labeled oligonucleotide. The roles of specific residues in DNA binding were assessed by stoichiometric titration of d(A)6, which indicated for instance that Phe43 forms base stacking interactions with the single-stranded DNA. Finally, all results were combined to form a set of experimental restraints, which were subsequently used in restrained molecular dynamics calculations aimed at building a model for the Pf3 nucleoprotein complex. Implying in addition some similarities to the well-studied M13 complex, a plausible model could be constructed that is in accordance with the experimental data.

A mutant of bacteriophage M13 was prepared in which a cysteine residue was introduced at position 25 of the major coat protein. The mutant coat protein was spin-labeled with a nitroxide derivative of maleimide and incorporated at different lipid-to-protein (L/P) ratios in DOPC or DOPG. The rotational dynamics of the reconstituted mutant coat protein was studied using EPR and saturation transfer (ST) EPR techniques. The spectra are indicative for an anisotropic motion of the maleimide spin label with a high order parameter (S = 0.94). This is interpreted as a wobbling motion of the spin label with a correlation time of about 10(-6) to 10(-5) s within a cone, and a rotation of the spin label about its long molecular axis with a correlation time of about l0(-7) s. The wobbling motion is found to correspond generally to the overall rotational motion of a coat protein monomer about the normal to the bilayer. This motion is found to be sensitive to the temperature and L/P ratio. The high value of the order parameter implies that the spin label experiences a strong squeezing effect by its local environment, that reduces the amplitude of the wobbling motion. This squeezing effect is suggested to arise from a turn structure in the coat protein from Gly23 to Glu20.

The backbone dynamics of the major coat protein (gVIIIp) of the filamentous bacteriophage M13, solubilized in detergent micelles, have been studied using 15N nuclear magnetic resonance spectroscopy at three frequencies. Motional parameters and overall and internal correlation times were derived with the model-free approach. It was also checked whether these parameters had to be modified due to anisotropic motion of the protein/micelle complex. Reduced spectral density mapping was used to calculate the spectral densities at J(O), J(omegaN), and [J(omegaH)]. The spectral densities were interpreted by mapping a linear or scaled linear combination of two Lorentzians onto a J(O)-J(omega) plot. The major coat protein of bacteriophage M13 consists of two alpha-helices, one of which is hydrophobic and located within the micelle, while the other is amphipathic and located on the surface of the micelle. Our results indicate that the motion of the hydrophobic helix is restricted such that it corresponds to the overall tumbling of the protein/micelle complex. The interpretation of the relaxation data of the amphipathic helix by means of the model-free approach and the reduced spectral density mapping indicate that in addition to the overall motion all residues in this helix are subject to motion on the fast nanosecond and picosecond time scales. The motions of the vectors in the low nanosecond range are characterized by similar values of the spectral densities and correlation times and represent the motion of the amphipathic helix on and away from the surface of the micelle. The relaxation data of the residues in the hinge region connecting the helices show that there is an abrupt change from highly restricted to less restricted motion. Both the C-terminal and N-terminal residues are very mobile.

We report a floating chirality procedure to treat nonstereospecifically assigned methylene or isopropyl groups in the calculation of protein structures from NMR data using restrained molecular dynamics and simulated annealing. The protocol makes use of two strategies to induce the proper conformation of the prochiral centres: explicit atom 'swapping' following an evaluation of the NOE energy term, and atom 'floating' by reducing the angle and improper force constants that enforce a defined chirality at the prochiral centre. The individual contributions of both approaches have been investigated. In addition, the effects of accuracy and precision of the interproton distance restraints were studied. The model system employed is the 18 kDa single-stranded DNA binding protein encoded by Pseudomonas bacteriophage Pf3. Floating chirality was applied to all methylene and isopropyl groups that give rise to non-degenerate NMR signals, and the results for 34 of these groups were compared to J-coupling data. We conclude that floating stereospecific assignment is a reliable tool in protein structure calculation. Its use is beneficial because it allows the distance restraints to be extracted directly from the measured peak volumes without the need for averaging or adding pseudoatom corrections. As a result, the calculated structures are of a quality almost comparable to that obtained with stereospecific assignments. As floating chirality furthermore is the only approach treating prochiral centres that ensures a consistent assignment of the two proton frequencies in a single structure, it seems to be preferable over using pseudoatoms or (R(-6)) averaging.

The dynamics of the backbone and (some of) the side chains of lac headpiece (1-56; lac HP56) have been studied for the free protein and for its complex with lac half-operator DNA by 15N T1 and T1p relaxation measurements combined with [1H-15N] NOE experiments. For the structurally well-defined part of the free lac HP56 (i.e., residues 3-49) a rigid backbone was found for residues in the three alpha-helices and for the turn of the helix-turn-helix motif. The loop between helices II and III of lac headpiece, which was characterized by slight disorder in the structure calculations, shows increased mobility. The detected side chains are very mobile. These data are in full agreement with the rms deviations in the structural data of free lac HP56. When lac HP56 is complexed with DNA, several changes in mobility take place. The most remarkable change was found for the loop region between helices II and III: residue His-29 within this loop interacts with Thy-3 of the operator DNA. As a result this mobile loop adapts itself to the DNA and becomes more rigid. Moreover, most DNA-contacting side chains show a significant decrease in flexibility, although these side chains do not become as rigid as the backbone. These results suggest that the mobility of the regions within lac HP56 important for complexation, i.e., the loop and the DNA-contacting side chains, is essential for a good fit onto the counterparts of the target DNA.

The lantibiotic nisin is a cationic, polycyclic bacteriocin of 34 residues, including several unusual dehydro residues and thioether-bridged lanthionines. The primary target of its antimicrobial action is the cytoplasmic membrane. Therefore the conformation of nisin when bound to membrane-mimicking micelles of zwitterionic dodecylphosphocholine and of anionic sodium dodecylsulphate was determined with high-resolution NMR spectroscopy. Structures were calculated on the basis of NMR-derived constraints with the distance-geometry program DIANA and were further refined by restrained energy minimization using X-PLOR. The conformation of nisin complexed to both types of micelles is the same, irrespective of the different polar head-groups of the detergents. The structure consists of two structured domains: an N-terminal domain (residues 3-19) containing three lanthionine rings, A, B and C; and a C-terminal domain (residues 22-28) containing two intertwined lanthionine rings numbered D and E. These domains are flanked by regions showing structural variability. Both domains are clearly amphipathic, a property characteristic for membrane-interacting polypeptides. The structures of the ring systems are better defined than those of the linear segments. The four-residue rings B, D and E of nisin all show a beta-turn structure, which is closed by the thioether linkage. The backbones of the rings B and D form type 11 beta-turns. Ring E resembles a type I beta-turn. Preceding the intertwined rings D (residues 23-26) and E (25-28) another type-II beta-turn is found formed by the residues 21-24, so that the C-terminal domain consists of three consecutive beta-turns. The structures of nisin in the micellar systems differ significantly from the previously determined (and now partially recalculated) structure in aqueous solution [van de Ven, F. J. M., van den Hooven, H. W., Konings, R. N. H. & Hilbers, C. W. (1991) Eur J. Biochem. 202, 1181-1188] in the first lanthionine ring around dehydroalanine 5.

The interaction of nisin, a membrane-interacting cationic polypeptide, with membrane-mimicking micelles of zwitterionic dodecylphosphocholine and of anionic sodium dodecylsulphate was studied. Direct contacts have been established through the observation of NOEs between nisin and micelle protons. Spin-labeled DOXYL-stearic acids were incorporated into the two micellar systems. From the paramagnetic broadening effects induced in the 1H-NMR spectrum of nisin it is concluded that the molecule is localized at the surface of the micelles. The interactions of nisin with zwitterionic and with anionic micelles resemble each other as do the nisin conformations [van den Hooven, H. W., Doeland, C. C. M., van de Kamp, M., Konings, R. N. H., Hilbers, C. W. & van de Ven, F. J. M. (1995) Eur J. Biochem. 235, 382-393]. The hydrophobic residues are immersed into the micelles and oriented towards the center, whereas the more polar or charged residues have an outward orientation. The micellar systems are considered to model the first step in the mechanism of antimicrobial action of nisin, this step is the binding of nisin to the cytoplasmic membrane of target bacteria. Detailed information on this initial binding step is obtained. Hydrophobic and electrostatic interactions appear to be involved in the nisin-micelle contacts. It is suggested that subtilin, a lantibiotic structurally related to nisin, has a comparable membrane interaction surface.

The membrane-bound form of the major coat protein (gVIIIp) of bacteriophage M13 has been studied using nuclear magnetic resonance spectroscopy. As membrane mimetics, we used dodecylphosphocholine (DodPCho) detergent micelles to solubilize the protein. We were able to nearly completely assign all resonances of the protein solubilized in DodPCho micelles by using both homonuclear and heteronuclear multidimensional experiments. Based on the patterns of the nuclear Overhauser enhancements and the chemical shifts of the resonances, we deduced the secondary structure of the protein. Additional structural information was obtained from amide proton exchange data and J-coupling constants. The protein consists of two alpha-helices which are connected by a hinge region around residue 21. From spin-label experiments, the location of the protein relative to the DodPCho micelles was determined. One, hydrophobic, helix spans the micelle, and another, amphipathic, helix, is located beneath the surface of the micelle. Comparison of the data of gVIIIp in DodPCho micelles with those of gVIIIp in sodium dodecyl sulfate (SDS) micelles [Van de Ven, F. J. M., van Os, J. W. M., Aelen, J. M. A., Wymenga, S. S., Remerowski, M. L., Konings, R. N. H. & Hilbers, C. W. (1993) Biochemistry 32, 8322-8328; Papavoine, C. H. M., Konings, R. N. H., Hilbers, C. W. & Van de Ven, F. J. M. (1994) Biochemistry 33, 12,990-12,997] reveals that the structures of the protein in the two detergent micelles are very similar. They differ only in the arrangement of the detergent molecules around the protein. For gVIIIp in SDS micelles, we found a micellar structure which is distorted near the C-terminus of the protein; whereas for DodPCho micelles, both distorted and regular elliptical micelles occur. This distortion is probably due to the interaction of the positively charged lysine side chains with the negatively charged head group of the detergent molecules.

The three-dimensional solution structure of mutant Tyr41-->His of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been refined in two stages. The first stage involved the collection of additional NOE-based distance constraints, which were then used in eight cycles of back-calculations and structure calculations. The structures of the gene V protein dimers were calculated using simulated annealing, employing restrained molecular dynamics with a geometric force field. In the second stage of the refinement procedure, solvent was explicitly included during the dynamic calculations. A total of 30 structures was calculated for the protein, representing its solution structure in water. The first calculation step significantly improved the convergence of the structures, whereas the subsequent simulations in water made the structures physically more realistic. This is, for instance, illustrated by the number of hydrogen bonds formed in the molecule, which increased considerably upon going to aqueous solution. It is shown that the solution structure of the mutant gene V protein is nearly identical to the crystal structure of the wild-type molecule, except for the DNA-binding loop (residues 16-28). This antiparallel beta-hairpin is twisted and partially folded back towards the core of the protein in the NMR structure, whereas it is more extended and points away from the rest of the molecule in the X-ray structure. Unrestrained molecular dynamics calculations suggest that this latter conformation is energetically unstable in solution.

The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins.

A (1)H NOESY experiment with two (13)C half-filters is described which has, compared to previously reported versions, an enhanced overall sensitivity and strongly reduced intramolecular cross peaks in any part of the spectrum edited for intermolecular NOEs. By adding a shaped (13)C pulse to the half-filter which selectively inverts the aromatic resonances, the filter can be tuned separately and simultaneously for the aliphatic and aromatic regions. Contrary to recently proposed schemes, no magnetization is destroyed, so that full sensitivity is retained for symmetric systems such as homodimers. Furthermore, by replacing the rectangular 180° (13)C pulses by high-power hyperbolic secant pulses for inversion of the complete (13)C spectral range, offset effects (which are another source of signal loss and artefacts) are eliminated. The spectra edited for intermolecular NOEs clearly demonstrate that residual artefacts are considerably smaller than in the original version of the experiment.

Lantibiotics are bacteriocins that contain unusual amino acids such as lanthionines and alpha, beta-didehydro residues generated by posttranslational modification of a ribosomally synthesized precursor protein. The structural gene encoding the novel lantibiotic epilancin K7 from Staphylococcus epidermidis K7 was cloned and its nucleotide sequence was determined. The gene, which was named elkA, codes for a 55-residue preprotein, consisting of an N-terminal 24-residue leader peptide, and a C-terminal 31-residue propeptide which is posttranslationally modified and processed to yield mature epilancin K7. In common with the type-A lantibiotics nisin A and nisin Z, subtilin, epidermin, gallidermin and Pep5, pre-epilancin K7 has a so-called class-Al leader peptide. Downstream and upstream of the elkA gene, the starts of two open-reading-frames, named elkP and elkT, were identified. The elkP and elkT genes presumably encode a leader peptidase and a translocator protein, respectively, which may be involved in the processing and export of epilancin K7. The amino acid sequence of the unmodified pro-epilancin K7, deduced from the elkA gene sequence, is in full agreement with the amino acid sequence of mature epilancin K7, determined previously by means of NMR spectroscopy [van de Kamp, M., Horstink, L. M., van den Hooven, M. W., Konings, R. N. M., Hilbers, C. W., Sahl, H.-G., Metzger, J. W. & van de Ven, F. J. M. (1995) Eur. J. Biochem. 227, 757-771]. The first residue of mature epilancin K7 appears to be modified in a way that has not been described for any other lantibiotic so far. NMR experiments show that the elkA-encoded serine residue at position +1 of pro-epilancin K7 is modified to a 2-hydroxypropionyl residue in the mature protein.

Nisin is a cationic polycyclic bacteriocin secreted by some lactic acid bacteria. Nisin has previously been shown to permeabilize liposomes. The interaction of nisin was analyzed with liposomes prepared of the zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG). Nisin induces the release of 6-carboxyfluorescein and other small anionic fluorescent dyes from PC liposomes in a delta psi-stimulated manner, and not that of neutral and cationic fluorescent dyes. This activity is blocked in PG liposomes. Nisin, however, efficiently dissipates the delta psi in cytochrome c oxidase proteoliposomes reconstituted with PG, with a threshold delta psi requirement of about -100 mV. Nisin associates with the anionic surface of PG liposomes and disturbs the lipid dynamics near the phospholipid polar head group-water interface. Further studies with a novel cationic lantibiotic, epilancin K7, indicate that this molecule penetrates into the hydrophobic carbon region of the lipid bilayer upon the imposition of a delta psi. It is concluded that nisin acts as an anion-selective carrier in the absence of anionic phospholipids. In vivo, however, this activity is likely to be prevented by electrostatic interactions with anionic lipids of the target membrane. It is suggested that pore formation by cationic (type A) lantibiotics involves the local perturbation of the bilayer structure and a delta psi-dependent reorientation of these molecules from a surface-bound into a membrane-inserted configuration.

The amino acid sequence of the novel lantibiotic epilancin K7 from Staphylococcus epidermidis K7 was determined by NMR spectroscopy. NMR spectroscopy was used because sequencing by conventional Edman degradation techniques was prohibited by internal sequence blocks owing to the presence of modified residues. Epilancin K7 consists of 31 residues, including two alpha,beta-didehydroalanine (one-letter code U) and two alpha,beta-didehydrobutyrine (O) residues, one lanthionine (A-S-A), two beta-methyllanthionines (A*-S-A), and six lysines. Epilancin K7 has a molecular mass of 3032 +/- 1.5 Da. The amino acid sequence of epilancin K7 was derived from both through-space dipolar proton-proton interactions and through-bond scalar proton-carbon interactions as detected by two-dimensional 1H-NOESY, 1H-ROESY and three-dimensional 1H-TOCSY-NOESY, and by two-dimensional 1H,13C-heteronuclear multiple-bond correlation spectroscopy, respectively. The sequence is as follows: [sequence: see text] The N-terminal residue X partly resembles an alanine but its exact nature is unclear. The organization of the sulfide-bridge-containing (beta-methyl-)lanthionines was revealed by 1H-NMR and 1H,13C-NMR spectroscopy. Epilancin K7 has a linear structure and a high positive net charge, and therefore is classified as a type-A lantibiotic. NMR analysis of a degraded though still active form of epilancin K7 showed that two N-terminal residues of epilancin K7 were missing, owing to decomposition at the alpha,beta-didehydro alanine at position 3; it was called the epilancin K7-(3-31)-peptide (peptide fragment of epilancin K7 consisting of positions 3-31). The usefulness of three-dimensional 1H-TOCSY-NOESY, and two-dimensional 1H,13C-heteronuclear multiple-bond correlation spectroscopy at natural abundance for the study of (modified) polypeptides is demonstrated.

Nuclear magnetic resonance spectroscopy was employed to study the single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3. The protein is 78 amino acids long and occurs in solution predominantly as a homodimer with a molecular mass of 18 kDa. Sequence-specific 1H and 15N resonance assignments have been obtained using homo- and heteronuclear two- and three-dimensional experiments. The secondary structure of the protein monomer was determined from a qualitative interpretation of nuclear Overhauser enhancement spectra and amide exchange data. It consists of a five-stranded antiparallel beta-sheet and three beta-hairpins. Problems caused by the protein's tendency to aggregate at concentrations needed for NMR spectroscopy were largely overcome by designing a mutant (Phe36-->His) which exhibits significantly improved solubility characteristics over the wild-type protein. It is shown that this mutation only locally affects the structure of the protein; the chemical shifts of the wild-type and mutant species differ only for a few residues near the site of the mutation, and the secondary structures of the proteins are identical. The secondary structure of the Pf3 single-stranded DNA binding protein is compared to that of the Ff gene V protein, the only single-stranded DNA binding protein for which the complete three-dimensional structure is known to date [Folkers, P. J. M., Nilges, M., Folmer, R. H. A., Konings, R. N. H. & Hilbers, C. W. (1994) J. Mol. Biol. 236, 229-246; Skinner, M. M., Zhang, H., Leschnitzer, D. H., Guan, Y., Bellamy, H., Sweet, R. M., Gray, C. W., Konings, R. N. H., Wang, A. H.-J. & Terwilliger, T. C. (1994) Proc. Natl Acad. Sci. USA 91, 2071-2075]. It is found that the secondary structures of the two proteins are very similar which supports the hypothesis that a five-stranded antiparallel beta-sheet with protruding beta-hairpins is a common motif in a certain class of single-stranded DNA binding proteins. In addition, the sequence and folding predicted earlier for the DNA binding wing in the single-stranded DNA binding protein of phage Pf3 [de Jong, E. A. M., van Duynhoven, J. P. M., Harmsen, B. J. M., Tesser, G. I., Konings, R. N. H. & Hilbers, C. W. (1989) J. Mol. Biol. 206, 133-156] is borne out by the present study. It closely resembles that in the single-stranded DNA binding protein of phage Ff, which may indicate that such a wing is a recurrent motif as well.

A contact analysis and a series of restrained molecular dynamics simulations were employed to derive a model of the complex between single-stranded DNA and the single-stranded DNA-binding protein encoded by gene V of the filamentous phage M13. The study is based on the recently elucidated solution structure of the Tyr41-->His mutant of the protein. Electron microscopy studies, indicating that the complex forms a flexible, left-handed helical coil with a diameter of 8 to 9 nm and an average pitch of 9 nm, were taken into consideration. The contact analysis served to determine the helix parameters that permit the energetically most favourable packing of protein molecules. Then a protein super-helix was built, into which two extended strands of DNA were modelled using restrained molecular dynamics. Specific constraints were included to ensure that the DNA would position itself into the binding groove of the protein. These constraints are based on recent NMR spin label experiments which offered a direct identification of the amino acids of the protein present in the DNA-binding domain. We present a model for the complex which is in full agreement with the existing reliable biophysical and biochemical data. A description of the protein-protein interface is given and the protein-DNA interaction is discussed in view of the derived model. In addition, we demonstrate that, on the basis of the available experimental data, and not imposing the left-handedness of the nucleoprotein complex, it is feasible to build also a plausible model for the complex which exhibits the opposite, i.e. right-handed, helical sense. This nucleoprotein structure features characteristics highly similar to those of the left-handed helix.

Gene V protein (GVP) encoded by the filamentous phage Ff (M13, fl, fd) is a homodimeric protein of 87 amino acids that binds to single-stranded DNA (ssDNA) nonspecifically and cooperatively. The structure (monoclinic C2 form) of the wild-type protein has been determined and refined at 1.8-A resolution [Skinner et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2071-2075]. The monomer structure consists of a somewhat distorted five-stranded beta-barrel core with three prominent loops: a DNA-binding loop, a dyad loop, and a dimer contact loop. The amino acid residue at position 41 plays an important role in the dimer-dimer interactions of the protein-ssDNA complex. Two Y41 mutant structures have been studied by X-ray crystallography. The Y41F GVP structure has been refined to an R-factor of 0.180 at 2.2-A resolution and is very similar to the wild-type (wt) structure (rmsd of all C alpha atoms = 0.30 A). In contrast, Y41H GVP forms a new crystal lattice in the space group P2(1)2(1)2(1) with a = 77.18 A, b = 84.17 A, and c = 28.62 A. Its structure has been solved by the molecular replacement method and refined to an R-factor of 0.170 at 2.5-A resolution. The two monomers of Y41H are crystallographically independent, and their structures remain similar to wt-GVP but with significant differences, particularly in the DNA-binding hairpin region. In both crystals, the loop (residues 36-43) that contains the Y41 residue is involved in the crystal dimer packings but in a different manner. The dimer-dimer contacts found in the wt-GVP crystal may be important for GVP aggregation in the absence of DNA. In the presence of DNA, the dimer-dimer contacts may switch to the type found in the Y41H crystal, allowing the GVP-ssDNA complex to form cooperatively. A model of the complex, consistent with existing biochemical and biophysical data, has been constructed from those crystal packing data.

A novel Plasmodium falciparum sporozoite antigen, STARP (Sporozoite Threonine and Asparagine-Rich Protein), detected consistently on the surface of sporozoites obtained from laboratory strains and field isolates, has been identified and cloned, following a systematic approach aimed at isolating novel non-CS sporozoite surface antigens. The 2.0-kb STARP gene has a 5' miniexon/large central exon structure and contains a complex repetitive region encoding multiple dispersed motifs and tandem 45- and 10-amino acid repeats. In sporozoites, transcription of the STARP gene has been conclusively demonstrated by reverse PCR and Northern blot hybridisation and the 78-kDa protein has been localized by immunofluorescence and immunoelectron microscopy to the sporozoite surface. STARP is also expressed in liver stages, as revealed by immunofluorescence assays using antisera raised either to the central repetitive region or the C-terminal non-repetitive region. Expression is also detected in early ring stages, though not in mature erythrocytic or sexual stages. Identification and elucidation of this novel antigen is a step forward in current efforts aimed at developing an effective preerythrocytic-stage malaria vaccine.

The solution structure of mutant Tyr41-->His of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been investigated by nuclear magnetic resonance spectroscopy. Two- and three-dimensional NMR experiments have been employed with a variety of NMR samples of gene V protein, some of which were uniformly enriched with either 15N or 13C. The structure of mutant Tyr41-->His of the M13 gene V protein which occurs in solution as a symmetric dimer was calculated using a two-stage procedure. The first step of the procedure involved the calculation of a set of individual monomer structures using the distance geometry program DIANA. This was then followed by the calculation of dimer structures employing "simulated annealing" protocols with the program X-PLOR. Hereby, the problem of assignment of intra- and inter-subunit NOEs of the symmetric dimer was circumvented through use of a target function that correctly deals with the intra- and inter-subunit contributions to the NOE peaks. Furthermore, a pseudo energy term was employed to restrain the symmetry of the dimer. In addition to this novel calculation strategy, we have incorporated distance information for a set of NOEs which were unambiguously identified as inter-subunit NOEs using an NMR strategy based on asymmetric labelling. A total of 20 structures were calculated for the M13 gene V protein mutant Tyr41-->His based on approximately 1000 experimental restraints derived from the NMR data. The structure of residues 1 to 15 and 29 to 87 of both monomers is reasonably well determined with an average atomic r.m.s. difference between the individual structures and the respective mean structure of approximately 0.9 A for the backbone atoms and approximately 1.4 A for all atoms. The orientation of the exposed anti-parallel beta-loop (residues 16 to 28) with respect to the core could not be determined. The molecular architecture of each of the monomers includes a five-stranded beta-barrel enclosing a hydrophobic core and two-antiparallel beta-loops. The dimer structure is stabilized predominantly by hydrophobic residues primarily involving the symmetry-related dyad domains (residues 64 to 82) of the monomers. Residues which are close to bound single-stranded DNA were identified previously from binding experiments with spin-labelled oligonucleotides. The solution structure of mutant Tyr41-->His of the M13 gene V protein is consistent with these binding data and provides a clear view of the protein's single-stranded DNA binding path.

The single-stranded DNA binding protein, or gene V protein (gVp), encoded by gene V of the filamentous bacteriophage M13 is a multifunctional protein that not only regulates viral DNA replication but also gene expression at the level of mRNA translation. It furthermore is implicated as a scaffolding and/or chaperone protein during the phage assembly process at the hostcell membrane. The protein is 87 amino acids long and its biological functional entity is a homodimer. In this manuscript a short description of the life cycle of filamentous phages is presented and our current knowledge of the major functional and structural properties and characteristics of gene V protein are reviewed. In addition models of the superhelical complexes gVp forms with ssDNA are described and their (possible) biological meaning in the infection process are discussed. Finally it is described that the 'DNA binding loop' of gVp is a recurring motif in many ssDNA binding proteins and that the fold of gVp is shared by a large family of evolutionarily conserved gene regulatory proteins.

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.

The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of Plasmodium falciparum has been cloned. The Pfs48/45 gene is a non-interrupted, single copy gene that codes for a hydrophobic, non-repetitive protein of 448 amino acid residues containing a putative signal peptide at the N-terminus, a hydrophobic C-terminus and 7 potential N-glycosylation sites. Antibodies directed against a Pfs48/45-glutathione-S-transferase fusion protein reacted with both the 45-kDa and 48-kDa proteins of gametocytes. When Pfs48/45 is expressed in the baculovirus-insect cell system the recombinant Pfs48/45 protein is targeted and exposed to the insect cell surface in such a configuration that it is recognized by transmission-blocking anti-45/48-kDa monoclonal antibodies.

Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.

The major coat protein (gVIIIp) of bacteriophage M13 complexed with SDS detergent micelles was used as a model system to study the lipid-bound conformation of the protein. Conditions were found that allowed the recording of good quality of NMR spectra. By making extensive use of three-dimensional heteronuclear (13C, 15N) NMR, we obtained a complete set of resonance assignments for 1HN, 1H alpha, 1H beta, 13C alpha, CO, and 15N and partially assigned the rest of the 1H spectrum. Analysis of NOE and chemical shift data reveals that gVIIIp is composed of two alpha-helical domains, one ranging from Pro-6 to Glu20 and the other ranging from Tyr-24 all the way to the C-terminus Ser-50. In contrast to the results reported by Henry and Sykes [Henry, G.D., & Sykes, B.D. (1992) Biochemistry 31, 5285-5297], at a high SDS to protein ratio the protein appears to be monomeric.

The lantibiotic, nisin, which is known to interact with membranes of certain Gram-positive bacteria, was studied in three model systems which mimic a membrane-like environment, i.e. a mixture of trifluoroethanol and water, or micelles of sodium dodecyl sulfate or dodecylphosphocholine. The 1H NMR spectra of nisin in the non-aqueous environments, at 40 degrees C and pH 3.5, have been assigned completely. The CD and NMR results indicate that the conformation of nisin in the three non-aqueous environments differs from that in aqueous solution, and that the conformation in the two micellar systems is similar. The major conformational changes, relative to nisin in aqueous solution, occur in the N-terminus.

This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions. The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values. In addition, all proteins measured bind a more-or-less co-operative manner to single-stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H greater than wild-type greater than F68L and R16H greater than Y41F and Y41H greater than F73L greater than R21C greater than Y34H greater than G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.