Design of an artificial phage-display library based on a new scaffold improved for average stability of the randomized proteins.

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the µM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.

Experimental observation of asymmetrical microwave jets and far-field distribution generated by a dual-material system.

In this paper, we report the experimental and numerical investigation of plane wave diffraction by an all-dielectric dual-material cuboid. Edge diffraction by a cuboid leads to the generation of a narrow, high intensity beam in the near-field region called a photonic jet. We examine the dependence of the jet behavior and orientation on the materials and dimensions of constitutive parts in the microwave frequency domain. The possibility to shift and deviate the resultant microwave jet in the near-field region of such a structure depending on the size of constitutive parts is demonstrated numerically. Experimentally, we observe a shift in the spatial position of the jet. The experimental asymmetric electric field profile observed in the far-field region is attributed to the input of multiple edge waves generated by the dual-material cuboid. The presented results may be scaled at different frequency bands such as optical frequencies for designing nanostructures enabling the focusing and deviation functionality and creation of new optical devices which would satisfy the needs of emerging nanophotonic applications.

Immunization of Baboons with Schistosoma mansoni Cercariae attenuated by gamma irradiation.

Studies on the efficacy of a vaccine against schistosomiasis in young baboons (Papio anubis) disclosed that immunization with Schistosoma mansoni cercariae attenuated by gamma irradiation induced significant protection against subsequent infection with normal, viable S. mansoni cercariae. Such immunization resulted in reduced worm burdens (70 percent) and egg excretion rates (82 percent). These results support immunization as a potential method for schistosomiasis control.

Retinoic acid, but not arsenic trioxide, degrades the PLZF/RARalpha fusion protein, without inducing terminal differentiation or apoptosis, in a RA-therapy resistant t(11;17)(q23;q21) APL patient.

Primary blasts of a t(11;17)(q23;q21) acute promyelocytic leukaemia (APL) patient were analysed with respect to retinoic acid (RA) and arsenic trioxide (As2O3) sensitivity as well as PLZF/RARalpha status. Although RA induced partial monocytic differentiation ex vivo, but not in vivo, As203 failed to induce apoptosis in culture, contrasting with t(15;17) APL and arguing against the clinical use of As203 in t(11;17)(q23;q21) APL. Prior to cell culture, PLZF/RARalpha was found to exactly co-localize with PML onto PML nuclear bodies. However upon cell culture, it quickly shifted towards microspeckles, its localization found in transfection experiments. Arsenic trioxide, known to induce aggregation of PML nuclear bodies, left the microspeckled PLZF/RARalpha localization completely unaffected. RA treatment led to PLZF/RARalpha degradation. However, this complete PLZF/RARalpha degradation was not accompanied by differentiation or apoptosis, which could suggest a contribution of the reciprocal RARalpha/PLZF fusion product in leukaemogenesis or the existence of irreversible changes induced by the chimera.

A "loop entropy reduction" phage-display selection for folded amino acid sequences.

As a step toward selecting folded proteins from libraries of randomized sequences, we have designed a 'loop entropy reduction'-based phage-display method. The basic premise is that insertion of a long disordered sequence into a loop of a host protein will substantially destabilize the host because of the entropic cost of closing a loop in a disordered chain. If the inserted sequence spontaneously folds into a stable structure with the N and C termini close in space, however, this entropic cost is diminished. The host protein function can, therefore, be used to select folded inserted sequences without relying on specific properties of the inserted sequence. This principle is tested using the IgG binding domain of protein L and the lck SH2 domain as host proteins. The results indicate that the loop entropy reduction screen is capable of discriminating folded from unfolded sequences when the proper host protein and insertion point are chosen.

Kinetic studies of the refolding of yeast phosphoglycerate kinase: comparison with the isolated engineered domains.

Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time-dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar-sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55-60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683-8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C-domain are masked during the refolding of yeast phosphoglycerate kinase. The N-domain was also found to refold faster when it was isolated than when integrated.

Key interactions in the immunoglobulin-like structure of apo-neocarzinostatin: evidence from nuclear magnetic resonance relaxation data and molecular dynamics simulations.

The three-dimensional structure of apo-neocarzinostatin (apo-NCS, MW: ca.11000, antitumoral chromophore carrier protein) is based on a seven-stranded antiparallel beta-sandwich, very similar to the immunoglobulin folding domain. We investigated the backbone dynamics of apo-NCS by (13)C-NMR relaxation measurements and molecular dynamics simulation. Model-free parameters determined from the experimental data are compared with a 1.5-nsec molecular simulation of apo-NCS in aqueous solution. This comparison provides an accurate description of both local and collective movements within the protein. This analysis enabled us to correlate dynamic processes with key interactions of this beta-protein. Local motions that could be relevant for the intermolecular association with the ligand are also described.

3 beta, 17 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni. Kinetic evidence for the bifunctional activity at a common catalytic site.

3 beta, 17 beta-Hydroxysteroid dehydrogenase (3 beta 17 beta HSDH) is an NAD-dependent dehydrogenase which has a double specificity for the 3- and 17-positions on the steroid skeleton. When dehydroepiandrosterone (DHEA) is used as steroid substrate, and the assay coupled with ketosteroid-isomerase, the two reactions occur alternately and each reaction on the 3-position produces a chromophoric molecule. These two reactions can follow one another without dissociation of the coenzyme from the enzyme binding site. This is confirmed by competition experiments with another dehydrogenase.

Cold denaturation of yeast phosphoglycerate kinase: which domain is more stable?

Under destabilising conditions both heat and cold denaturation of yeast phosphoglycerate kinase (PGK) can be observed. According to previous interpretation of experimental data and theoretical calculations, the C-terminal domain should be more stable than the N-terminal domain at all temperatures. We report on thermal unfolding experiments with PGK and its isolated domains, which give rise to a revision of this view. While the C-terminal domain is indeed the more stable one on heating, it reveals lower stability in the cold. These findings are of importance, because PGK has been frequently used as a model for protein folding and mutual domain interactions.

Flexibility and folding of phosphoglycerate kinase.

Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.

Reactivation by imidazo-pyridinium oximes of acetylcholinesterase inhibited by organophosphates. A study with an immobilized enzyme method.

A new procedure is described for studying inhibition and reactivation of acetylcholinesterase (AChE). The enzyme, electric eel AChE, is immobilized on fiberglass and the enzymatic activity is continuously monitored in an open reactor by an assay adapted from the Ellman's method. The use of immobilized AChE permits independent inhibition and reactivation of the enzyme. Side-reactions between substrate, inhibitor and reactivator are avoided. This method is used to determine the reactivating efficiency of a new series of imidazo-pyridinium oximes for the enzyme inhibited by different organophosphorous compounds. Kinetic parameters of reactivation were determined after AChE inhibition by sarin, VX and paraoxon. The more efficient reactivators have a short methylene bridge (C3 to C6) between imidazolium and pyridinium rings. Against soman inhibition, the pyrimidoxime or 1-(1-methyl-imidazolinium) 3-(4-carbaldoxime-pyridinium) propane dibromide, introduced immediately after the inhibitor, gives the same result as TMB-4 (37% reactivation). 1-benzyl 2-carbaldoxime pyridinium bromide was found to be more potent in reactivating tabun inhibited AChE than pyrimidoxime. Imidazo-pyridinium oximes with a long methylene bridge (C8 to C10) are good reversible inhibitors of free AChE (Ki less than 1 microM).

Proteolytic, antigenic and immunogenic properties of Schistosoma mansoni cercarial secretion material.

Schistosoma mansoni cercariae secrete preacetabular gland material in association with skin penetration. The material is heterogeneous and contains proteolytic enzyme(s) which comprise a small proportion of the total protein. We have immunized mice with cercarial secretion material in several protocols designed to induce a variety of antibody responses. The cercarial secretion material is sufficiently immunogenic to induce precipitating and reaginic antibody production. The antibodies obtained were not of the necessary quantity and/or quality to afford protection from a challenge infection.

Resistance of mice to secondary infection with Schistosoma mansoni. II. Evidence for a correlation between egg deposition and work elimination.

Mice reinfected with Schistosoma mansoni 6--8 weeks after a primary infection largely or completely eliminated the second infection prior to the 7-week adult worm stage. In contrast, challenge worm counts were not lower than controls at the 6-day lung schistosomulum stage. At reinfection intervals of 12 or more weeks, worm counts were reduced at both stages. The reduction in lung schistosomulum count was proportional to the number of schistosome eggs present in the lungs, with no significant reduction being detected at any challenge time in mice free of lung eggs. Isolated schistosome eggs injected intravenously into the lungs of normal mice induced moderate to high levels of resistance to infection, while eggs injected subcutaneously or imtraperitoneally did not. It is concluded that the deposition of schistosome eggs in sites encountered by migrating schistosomula may be essential for mice to become resistant to reinfection with S. mansoni.

Effect of immunization on migration of Schistosoma mansoni through lungs.

Schistosoma mansoni schistosomula migrate through the lungs of infected mice; peak recovery occurs 6 days following infection. When cercariae were irradiated at 60Cobalt doses of 24 to 64 Kr, the number of schistosomula recovered from lungs 6 days after infection decreased in a dose-dependent fashion. The pattern of lung migration of schistosomes irradiated at 56 Kr prior to infection was similar to that of nonirradiated schistosomes, but greatly reduced numbers were recovered. When mice were immunized by a single infection with 56 Kr attenuated cercariae 6 wk prior to challenge, the pattern of challenge migration through the lungs was altered. The number of schistosomes recovered increased rapidly between 2 and 6 days post-infection, and showed a plateau between 8 and 10 days with peak recovery occurring 10 days post-infection. This peak recovery was reduced in comparison to that of non-immunized mice, but did not account for all of the reduction observed at the adult worm stage.

Resistance of mice to secondary infection with Schistosoma mansoni. I. Comparison of bisexual and unisexual initial infections.

Mice receiving a unisexual primary infection with either sex of Schistosoma mansoni did not develop detectable resistance to reinfection. In contrast, mice receiving a bisexual primary infection developed a high degree of resistance. The number of adult worms developing from the challenge infection was reduced, relative to controls, by 72--100% at challenge times of 6 weeks or greater.

Immunization of mice with cobalt-60 irradiated Schistosoma mansoni cercariae.

Parameters of immunization of mice with 60Cobalt-irradiated Schistosoma mansoni cercariae are described and related to protection against subsequent challenge infection. Such immunization was found to be dependent on dose of irradiation, number of immunizing cercariae, and number and time course of infections. Low levels of resistance were obtained with low irradiation doses, in contrast to previous studies in mice. In general, resistance increased with increasing irradiation doses, up to approximately 48-56 Kr. Maximal resistance (70-80%) was induced by a single exposure to 250-500 cercariae, irradiated at a dose rate of 2 Kr/minute to a total dose of 56 Kr, administered percutaneously 4-6 wk prior to challenge. Challenge could be delayed for at least 15 wk after immunization without a decrease in resistance. The resistance obtained was not attributable to a delayed migration of challenge worms.

How random is a highly denatured protein?

There has been renewed interest in determining the physicochemical properties of denatured states of proteins. In many denatured states there is evidence for the existence of nonrandom configurational distributions. Here we examine the small-angle neutron scattering profile of yeast phosphoglycerate kinase in the native state and in highly denaturing conditions. We show that the denatured protein scattering profile can be interpreted using a model developed for synthetic polymers in which the chain behaves as a random coil in a good solvent, i.e. with excluded volume interactions. The implications of this result for our appreciation of the protein folding process are discussed.

Murine immunization by cesium-137 irradiation attenuated Schistosoma mansoni cercariae.

Cesium-137, becoming a more readily available ionizing gamma radiation source for laboratory use, was shown to effectively attenuate Schistosoma mansoni cercariae for vaccine production. In parallel comparison studies with the murine model, cesium-137 attenuated cercariae consistently afforded better (P greater than 0.05) protection than did the cobalt-60 prepared vaccine. Dose-response data indicated that the optimal total irradiation with cesium-137 was between 45 and 50 Krad.