Ligands turning around in the midst of protein conformers: the origin of ligand-protein mating. A NMR view.

Protein-ligand binding is a puzzling process. Many theories have been devised since the pioneering key-and-lock hypothesis based on the idea that both the protein and the ligand have a rigid single conformation. Indeed, molecular motion is the essence of the universe. Consequently, not only proteins are characterized by an extraordinary conformational freedom, but ligands too can fluctuate in a rather vast conformational space. In this scenario, the quest to understand how do they match is fascinating. Recognizing that the inherent dynamics of molecules is the key factor controlling the success of the binding and, subsequently, their chemical/biological function, here we present a view of this process from the NMR stand point. A description of the most relevant NMR parameters that can provide insights, at atomic level, on the mechanisms of protein-ligand binding is provided in the final section.

Positive inotropic effects of Tityus cambridgei and T. serrulatus scorpion venoms on skeletal muscle.

Toxins that block voltage-dependent K+ channels and those that modify Na+ channel gating exhibit positive inotropic effect on skeletal muscle. We compared the effect of the venom of Tityus cambridgei (Tc) and Tityus serrulatus (Ts) scorpions on mouse diaphragm force, in vitro. In indirect and direct (using D-tubocurarine 7.3 microM) stimulation, Tc, 10microg/mL, increased the contractile force, an effect prevented by tetrodotoxin (TTX) while Ts, 0.5 microg/mL, potentiated only indirectly stimulated diaphragm, thus indicating its activity is mainly mediated through acetylcholine release from nerve terminal. This effect is prevented by TTX and attenuated by the K+ channel opener cromakalim. In conclusion, our data show that while the positive inotropic effect of both venoms appears associated to the activity of Na+ and K+ channels, only Tc venom acts also directly on skeletal muscle. This finding call for further studies on Tc venom to identify the toxin responsible for its direct inotropic activity as it may have clinical applications.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.

Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein Molecular, functional, and immunoprotection analysis

The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His6-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant â-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20> rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.

PcF protein from Phytophthora cactorum and its recombinant homologue elicit phenylalanine ammonia lyase activation in tomato.

The phytotoxic protein PcF (Phytophthora cactorum-Fragaria) is a 5.6-kDa cysteine-rich, hydroxyproline-containing protein that is secreted in limited amounts by P. cactorum, an oomycete pathogen of tomato, strawberry and other relevant crop plants. Although we have shown that pure PcF triggers plant reactivity, its mechanism of action is not yet understood. Here we show that PcF, like other known fungal protein elicitors involved in pathogen-plant interaction, stimulates the activity of the defense enzyme phenylalanine ammonia lyase (EC 4.3.1.5) in tomato seedlings. Recognizing that a key step in understanding the mechanism of action of PcF at a molecular level is knowledge of its three-dimensional structure, we overexpressed this protein extracellularly in Pichia pastoris. The preliminary structural and functional characterization of a recombinant PcF homologue, N4-rPcF, is reported. Interestingly, although N4-rPcF is devoid of proline hydroxylation and has four additional amino acid residues attached to its N terminus, its secondary structure and biological activity are indistinguishable from wild-type PcF.

The X-ray structure of a recombinant major urinary protein at 1.75 A resolution. A comparative study of X-ray and NMR-derived structures.

Major urinary proteins belong to the lipocalin family and are present in the urine of rodents as an ensemble of isoforms with pheromonal activity. The crystal structure of a recombinant mouse MUP (rMUP) was solved by the molecular-replacement technique and refined to an R factor and R(free) of 20 and 26.5%, respectively, at 1.75 A resolution. The structure was compared with an NMR model and with a crystallographic structure of the wild-type form of the protein. The crystal structures determined in different space groups present significantly smaller conformational differences amongst themselves than in comparison with NMR models. Some, but not all, of the conformational differences between the crystal and solution structures can be explained by the influence of crystallographic contacts. Most of the differences between the NMR and X-ray structures were found in the N-terminus and loop regions. A number of side chains lining the hydrophobic pocket of the molecule are more tightly packed in the NMR structure than in the crystallographic model. Surprisingly, clear and continuous electron density for a ligand was observed inside the hydrophobic pocket of this recombinant protein. Conformation of the ligand modelled inside the density is coherent with the results of recent NMR experiments.

Determining the binding capability of the mouse major urinary proteins using 2-naphthol as a fluorescent probe.

The mouse major urinary proteins (MUPs) are an ensemble of isoforms secreted by adult male mice and involved in sexual olfactory communication. MUPs belong to the lipocalin superfamily, whose conserved structure is a beta-barrel made of eight antiparallel beta-strands forming a hydrophobic pocket that accommodates small organic molecules. A detailed knowledge of the molecular mechanism associated to the binding of those molecules can guide protein engineering to devise mutated proteins where the ligand specificity, binding affinity, and release rate can be modulated. Proteins with such peculiar properties may have interesting biotechnological applications for pest control, as well as in food and cosmetic industries. In this work, we demonstrate that the fluorescent molecule 2-naphthol binds to the natural ligand's binding site of MUPs with high affinity. In addition, we show that 2-naphthol binds to MUPs in its protonated form, that its fluorescence is blue-shifted, and the quantum yield is increased, thus confirming the high hydrophobicity of the protein pocket and the absence of proton acceptors inside the binding site. At large the results presented, besides demonstrating that the use of 2-naphthol provides a convenient and quick method for testing MUPs binding activity and to ascertain the quality of the protein preparation, suggest that MUPs can represent an interesting system for studying the photophysical characteristics of fluorescent molecules in a highly hydrophobic environment.

Solution structure of a recombinant mouse major urinary protein.

Major urinary proteins (MUPs) form an ensemble of protein isoforms which are expressed and secreted by sexually mature male mice only. They belong to the lipocalin superfamily and share with other members of this family the capacity to bind hydrophobic molecules, some of which are odorants. MUPs, either associated with or free of their natural ligands, play an important role in the reproductive cycle of these rodents by acting as pheromones. In fact, they are able to interact with receptors in the vomeronasal organ of the female mice, inducing hormonal and physiological responses by an as yet unknown mechanism. In order to investigate the structural and dynamical features of these proteins in solution, one of the various wild-type isoforms (rMUP: 162 residues) was cloned and subsequently isotopically labeled. The complete 1H, 13C and 15N resonance assignment of that isoform, achieved by using a variety of multidimensional heteronuclear NMR experiments, has been reported recently. Here, we describe the refined high-resolution three-dimensional solution structure of rMUP in the native state, obtained by a combination of distance geometry and energy minimization calculations based on 2362 NOE-derived distance restraints. A comparison with the crystal structure of the wild-type MUPs reveals, aside from minor differences, a close resemblance in both secondary structure and overall topology. The secondary structure of the protein consists of eight antiparallel beta-strands forming a single beta-sheet and an alpha-helix in the C-terminal region. In addition, there are several helical and hairpin turns distributed throughout the protein sequence, mostly connecting the beta-strands. The tertiary fold of the beta-sheet creates a beta-barrel, common to all members of the lipocalin superfamily. The shape of the beta-barrel resembles a calyx, lined inside by mostly hydrophobic residues that are instrumental for the binding and transport of small nonpolar ligand molecules.

Multicentre magnetic resonance texture analysis trial using reticulated foam test objects.

Texture analysis in magnetic resonance imaging has the ability to provide useful diagnostic information with respect to the discrimination of disease states of a single tissue or the separation of different tissues. However, for widespread use it is necessary to determine how texture measurements carried out in one center relate to those carried out in another. To this end, a multicentre trial has been performed where reticulated foam test objects have been scanned in six European centers according to a fixed protocol. It has been concluded that texture measurements are not transportable between centers. Principal component models calculated from the texture parameters collected in one center do not fit the data collected in another. Further trials are to investigate whether the reticulated foam test objects may be used to normalize tissue texture data collected in different centers.

Trypanosoma cruzi: conformational preferences of antigenic peptides bearing the immunodominant epitope of the B13 antigen.

The Trypanosoma cruzi recombinant protein B13 contains tandemly repeated domains and shows high sensitivity in the serological diagnosis of Chagas' disease. It has been shown that the immunodominant epitope of B13 is contained in the GDKPSLFGQAAAGDKPSLF-NH(2) sequence and that the hexapeptide AAAGDK seems to be the "core" of that epitope. Three peptides containing that "core" sequence, one corresponding to the entire repeat motif GDKPSLFGQAAAGDKPSLF-NH(2), pB13, and two smaller fragments, FGQAAAGDK-NH(2), S4, and QAAAGDKPS-NH(2), S5, have been tested in competitive ELISA with recombinant protein B13 in the solid phase against 40 chagasic sera from Brazilian patients. The median percentage inhibition for pB13, S4, and S5 were, respectively, 91, 86, and 68%. The possibility that the distinct antigenic activity of those peptides correlates with the existence of preferential conformational properties has been investigated by CD and NMR spectroscopy. Results indicate their propensity to adopt a helical configuration, centered in the AAAGDK sequence, and whose extent and stability directly correlates with the peptides' antigenicity. The data are discussed in the light of the existence of conformational preferences involving immunodominant epitopes in tandemly repeated antigens.

Structure of two fragments of the third cytoplasmic loop of the rat angiotensin II AT1A receptor. Implications with respect to receptor activation and G-protein selection and coupling.

The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT1A receptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the entire i3 loop and shown to differently interact with purified G-proteins have been obtained by simulated annealing calculations, using NMR-derived constraints collected in 70% water/30% trifluoroethanol solution. While the NH2-terminal half, Ni3, residues 213-231, adopts a stable amphipathic alpha-helix, extending over almost the entire peptide, a more flexible conformation is found for the COOH-terminal half, Ci3, residues 227-242. For this peptide, a cis-trans isomerization around the Lys6-Pro7 peptide bond generates two exchanging isomers adopting similar conformations, with an alpha-helix spanning from Asn9 to Ile15 and a poorly defined NH2 terminus. A quite distinct structural organization is found for the sequence EIQKN, common to Ni3 and Ci3. The data do suggest that the extension and orientation of the amphipathic alpha-helix, present in the proximal part of i3, may be modulated by the distal part of the loop itself through the Pro233 residue. A molecular model where this possibility is considered as a mechanism for G-protein selection and coupling is presented.

Structure of the C-terminal fragment 300-320 of the rat angiotensin II AT1A receptor and its relevance with respect to G-protein coupling.

Angiotensin II AT1A receptor is coupled to G-protein, and the molecular mechanism of signal transduction is still unclear. The solution conformation of a synthetic peptide corresponding to residues 300-320 of the rat AT1A receptor, located in the C-terminal cytoplasmic tail and indicated by mutagenesis work to be critical for the G-protein coupling, has been investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) and restrained molecular dynamics calculations. The CD data indicate that, in acidic water, at concentration below 0.8 mM, the peptide exists in a predominantly coil structure while at higher concentration it can form helical aggregates; addition of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. Using NMR-derived constraints, an ensemble of conformers for the peptide has been determined by restrained molecular dynamics calculations. Analysis of the converged three-dimensional structures indicates that a significant population of them adopts an amphipathic alpha-helical conformation that, depending upon experimental conditions, presents a variable extension in the stretch Leu6-Tyr20. An equilibrium with nonhelical structured conformers is also observed. We suggest that the capability of the peptide to modulate its secondary structure as a function of the medium dielectric constant, as well as its ability to form helical aggregates by means of intermolecular hydrophobic interactions, can play a significant role for G-protein activation.

Expression of a lipocalin in Pichia pastoris: secretion, purification and binding activity of a recombinant mouse major urinary protein.

The proteins of the mouse major urinary protein complex (MUP), members of the lipocalin family, bind volatile pheromones and interact with the vomeronasal neuroepithelium of the olfactory system. We report the expression of a MUP protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. Mature recombinant MUP (rMUP) is secreted at a concentration of 270 mg/l in minimal medium and it is isolated from the culture supernatant by one step ion-exchange chromatography in a nearly pure form. Binding activity, tested with an odorant molecule which displays high affinity for native MUP, indicates that rMUP has a behavior similar to the native one. This finding suggests that the protein, and in particular its hydrophobic binding pocket, is properly folded.

2-Naphthol-phosphatidylethanolamine: A fluorescent phospholipid analogue for excited-state proton transfer studies in membranes.

The fluorescence properties of the phospholipid derivative,N-[1-(2-naphthol)]-phosphatidylethanolamine (NAPH-PE), have been studied by steady-state and time-resolved fluorescence techniques. The new probe is a naphthol adduct of phosphatidylethanolamine. The emission spectrum of the fluorescent phospholipid depends on the pH and on the proton acceptor concentration as expected for a typical two-state excited-state proton transfer reaction. In ethanol solutions at an apparent pH of 6.7 and in the presence of acetate anion (0.14M), a biexponential decay is obtained from global analysis of the data. The lifetimes,τ 1=3.9 ns andτ 2=6.2 ns. are constant across the spectral region 350-460 nm. The decay-associated spectra and the species-associated spectra reproduce well the profiles reported for a two-state excited-state proton transfer reaction. The fluorescent phospholipid has been incorporated into dimyristoyllecithin and dipalmitoyllecithin vesicles. Although lower proton transfer is found, the reaction appears to be dependent on the gel-to-liquid-crystalline phase transition of the lipid membrane. In addition, the steady-state anisotropy of NAPH-PE measured as a function of temperature trace the phase transition of the two vesicle systems. Thus, it is shown that the physical state of the bilayer affects a reaction which takes place at the membrane surface. In the presence of acetate ions (0.3M), global analysis, performed in terms of fluorescence decay parameters, recovers preexponential coefficients that are consistent with an excited-state proton transfer reaction. The short lifetime drops from 3.9 to 0.44 ns without significant changes of the longer-lifetime component.