PubMed Health⌕ Search

Biomedical subjects

P Sodano

Publications and source records attributed to P Sodano.

At least 19 recordsLinked to original sources
PubMed2000

The active site of drosomycin, a small insect antifungal protein, delineated by comparison with the modeled structure of Rs-AFP2, a plant antifungal protein.

Drosomycin is the first strictly antifungal protein isolated from an insect (Drosophila melanogaster). The solution structure of this 44-residue protein has been reported previously. It involves a three-stranded beta-sheet and an alpha-helix, the protein global fold being maintained by four disulfide bridges. Rs-AFP2 is a plant antifungal protein exhibiting 41% sequence similarity with drosomycin. Mutational analysis of Rs-AFP2 showed the importance of some residues in the antifungal activity of the protein against the fungus target. In order to determine the structural features responsible for antifungal activity in both drosomycin and Rs-AFP2, we modeled the three-dimensional structure of Rs-AFP2, and of other antifungal proteins, using the solution structure of drosomycin as a template. Structure analysis of drosomycin and Rs-AFP2, and comparisons with the other modeled antifungal structures, revealed that the two proteins shared a hydrophobic cluster located at the protein surface in which a lysine residue is embedded. Based on these close structural similarities and the experimental data available for Rs-AFP2 mutants, an antifungal active site of the insect protein is proposed.

Amino Acid Sequence↗
PubMed1999

Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules.

Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular, NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane.

Acetonitriles↗
PubMed1999

Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins.

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns-LTP), a 91 amino acid residue protein belonging to the broad family of plant ns-LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side-chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H-1H Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution structure involves four helices (H1: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no observable regular structure. N-capping residues (Thr2, Ser24, Thr40), whose side-chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of alpha and 3(10) conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 +/- 0.16 A for all heavy atoms (C, N, O and S) and 0.72 +/- 0.01 A for the backbone atoms. The global fold of rice ns-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns-LTP. The sequential alignment of 36 ns-LTP primary structures strongly suggests that there is a uniform pattern of specific long-range interactions (in terms of sequence), which stabilize the fold of all plant ns-LTPs.

Amino Acid Sequence↗
PubMed1998

Solution structure of thanatin, a potent bactericidal and fungicidal insect peptide, determined from proton two-dimensional nuclear magnetic resonance data.

Thanatin is the first inducible insect peptide that has been found to have, at physiological concentrations, a broad range of activity against bacteria and fungi. Thanatin contains 21 amino acids including two cysteine residues that form a disulfide bridge. Two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling have been used to determine its three-dimensional (3D) structure in water. Thanatin adopts a well-defined anti-parallel beta-sheet structure from residue 8 to the C-terminus, including the disulfide bridge. In spite of the presence of two proline residues, there is a large degree of structural variability in the N-terminal segment. The structure of thanatin is quite different from the known structures of other insect defence peptides, such as antibacterial defensin and antifungal drosomycin. It has more similarities with the structures of various peptides from different origins, such as brevinins, protegrins and tachyplesins, which have a two-stranded beta-sheet stabilized by one or two disulfide bridges. Combined with activity test experiments on several truncated isoforms of thanatin, carried out by Fehlbaum et al. [Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J. P., Roussel, J. P., Letellier, L., Hétru, C. & Hoffmann, J. (1996) Proc. Natl Acad. Sci. USA 93, 1221-1225], our structural study evidences the importance of the beta-sheet structure and also suggests that anti-Gram-negative activity involves a site formed by the Arg20 side-chain embedded in a hydrophobic cluster.

Amino Acid Sequence↗
PubMed1998

Comparison of solution and crystal structures of maize nonspecific lipid transfer protein: a model for a potential in vivo lipid carrier protein.

The three-dimensional solution structure of maize nonspecific lipid transfer protein (nsLTP) obtained by nuclear magnetic resonance (NMR) is compared to the X-ray structure. Although both structures are very similar, some local structural differences are observed in the first and the fourth helices and in several side-chain conformations. These discrepancies arise partly from intermolecular contacts in the crystal lattice. The main characteristic of nsLTP structures is the presence of an internal hydrophobic cavity whose volume was found to vary from 237 to 513 A3 without major variations in the 15 solution structures. Comparison of crystal and NMR structures shows the existence of another small hollow at the periphery of the protein containing a water molecule in the X-ray structure, which could play an important structural role. A model of the complexed form of maize nsLTP by alpha-lysopalmitoylphosphatidylcholine was built by docking the lipid inside the protein cavity of the NMR structure. The main structural feature is a hydrogen bond found also in the X-ray structure of the complex maize nsLTP/palmitate between the hydroxyl of Tyr81 and the carbonyl of the lipid. Comparison of 12 primary sequences of nsLTPs emphasizes that all residues delineating the cavities calculated on solution and X-ray structures are conserved, which suggests that this large cavity is a common feature of all compared plant nsLTPs. Furthermore several conserved basic residues seem to be involved in the stabilization of the protein architecture.

Amino Acid Sequence↗
PubMed1998

Solution structure of Ace-AMP1, a potent antimicrobial protein extracted from onion seeds. Structural analogies with plant nonspecific lipid transfer proteins.

The three-dimensional solution structure of Ace-AMP1, an antifungal protein extracted from onion seeds, was determined using 1H NMR spectroscopy and molecular modeling. This cationic protein contains 93 amino acid residues and four disulfide bridges. Its structure was determined from 1260 NOE-derived distance restraints and 173 dihedral restraints derived from NOEs and 3JCaHNH coupling constants. The global fold involves four helical segments connected by three loops and a C-terminal tail without regular secondary structures, except for a 3(10)-helix turn and a beta-turn. The most striking feature is the absence of any continuous cavity running through the whole molecule as found in recently determined structures of nonspecific transfer proteins extracted from wheat and maize seeds, although their global folds are very similar. Consistent with the absence of a cavity in the core of Ace-AMP1, it was found that this protein, in contrast to ns-LTPs, does not bind fluorescently labeled phospholipids in solution. On the other hand, Ace-AMP1 is able to interact with phospholipid membranes as shown by the release of carboxyfluorescein from the lumen of artificial liposomes and by the induction of alterations in fluorescence polarization of fluorescently labeled phospholipids embedded in artificial liposomes.

Amino Acid Sequence↗
PubMed1998

A novel composite 90 degrees pulse sequence which provides distortionless NMR spectra and suppresses without destroying the water magnetization.

A novel 90 degrees composite pulse sequence which allows one to record 1D and 2D NMR spectra without disturbing the water magnetization is described. A home-written program was used to optimize the pulse angles for which the pulse sequence response fitted best the desired excitation profile, producing a neat and distortionless spectrum with a broad null excitation at the carrier frequency. The resulting pulse sequence was first evaluated using the simulation program "PENCIL" and then tested on two protein samples. A 3.5 degrees phase shift of the last pulse was required to cancel correctly the water signal. The pulse scheme was appended to a NOESY pulse sequence. Inspection of the water cross section revealed interactions between water and some protons of drosomycine, a small insect antifungal protein.

Amino Acids↗
PubMed1997

1H NMR and fluorescence studies of the complexation of DMPG by wheat non-specific lipid transfer protein. Global fold of the complex.

Plant non-specific lipid transfer proteins (LTPs) are proteins which transfer lipids between membranes in vitro and are believed to be involved in the transport of cutin monomers to the cuticle layer in vivo or in the plant defence against phytopathogens. The complexation of DMPG, a diacyl phospholipid, by wheat ns-LTP, a protein extracted from wheat seeds, was followed by 1H NMR and fluorescence spectroscopy. The global fold of the protein was calculated using the DIANA software package from a list of 968 distance constraints. The internal cavity volume, a feature common to all known ns-LTP structures, was estimated to be 750 A3 using the 'CAVITE' program. This model of the complex was obtained by inserting a lipid molecule in the cavity and was energy minimized. The study showed that the protein fold described for the free form was only weakly affected by the insertion of the bulky lipid. Observation of some intermolecular NOEs between the protein and the lipid glycerol moiety revealed that the cavity entrance was located between residues His35 and Arg44. The resulting solution structure was compared to the crystal structure of the maize ns-LTP/palmitate complex.

Arginine↗
PubMed1997

Refined solution structure of the anti-mammal and anti-insect LqqIII scorpion toxin: comparison with other scorpion toxins.

The solution structure of the anti-mammal and anti-insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatus was refined and compared with other long-chain scorpion toxins. This structure, determined by 1H-NMR and molecular modeling, involves an alpha-helix (18-29) linked to a three-stranded beta-sheet (2-6, 33-39, and 43-51) by two disulfide bridges. The average RMSD between the 15 best structures and the mean structure is 0.71 A for C alpha atoms. Comparison between LqqIII, the potent anti-mammal AaHII, and the weakly active variant-3 toxins revealed that the LqqIII three-dimensional structure is closer to that of AaHII than to the variant-3 structure. Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII. Several residues are well conserved in long-chain scorpion toxin sequences and seem to be important in protein structure stability and function. Some of them are involved in the CS alpha beta (Cysteine Stabilized alpha-helix beta-sheet) motif. A comparison between the sequences of the RII rat brain and the Drosophila extracellular loops forming scorpion toxin binding-sites of Na+ channels displays differences in the subsites interacting with anti-mammal or anti-insect toxins. This suggests that hydrophobic as well as electrostatic interactions are essential for the binding and specificity of long-chain scorpion toxins.

Amino Acid Sequence↗
PubMed1997

Solution structure of drosomycin, the first inducible antifungal protein from insects.

Drosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three-dimensional structure in aqueous solution was determined using 1H 2D NMR. This structure, involving an alpha-helix and a twisted three-stranded beta-sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized alpha beta (CS alpha beta) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short- and long-chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.

Amino Acid Sequence↗
PubMed1997

Homology modelling of an antimicrobial protein, Ace-AMP1, from lipid transfer protein structures.

BACKGROUND: Plant nonspecific lipid transfer proteins (ns-LTPs) are small basic proteins that facilitate lipid shuttling between membranes in vitro. The function of ns-LTPs in vivo is still unknown. It has been suggested, in relation to their lipid binding ability, that they may be involved in cutin formation. Alternatively, they may act in the plant defence system against pathogenic agents. Ace-AMP1 is an antimicrobial protein extracted from onion seed that shows sequence homology with ns-LTPs but that is unable to transfer lipids. We have recently determined the three-dimensional structure of wheat and maize ns-LTPs. In order to compare the structural features of Ace-AMP1 and ns-LTPs, we have used the comparative modelling software MODELLER to predict the structure of Ace-AMP1. RESULTS: The global fold of Ace-AMP1 is very similar to those of ns-LTPs, involving four helices and a C-terminal tail without secondary structure elements. The structure of maize and wheat ns-LTP is characterized by the existence of a tunnel-like hydrophobic cavity in which a lipid molecule can be inserted. In the Ace-AMP1 structure, this cavity is blocked by a number of bulky residues. Similarly, the electrostatic potential contours of ns-LTPs show some common features that were not observed in Ace-AMP1. CONCLUSIONS: Although Ace-AMP1 displays a similar global fold to ns-LTPs, it does not present a hydrophobic cavity, which may explain why Ace-AMP1 cannot shuttle lipids between membranes in vitro. The large differences in the electrostatic properties of Ace-AMP1 and ns-LTPs suggest a different mode of interaction with membranes.

Amino Acid Sequence↗
PubMed1996

Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds.

The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1,091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (phi, psi, chi 1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413-422) constitutes a new architecture for alpha-class proteins. 1H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 +/- 0.6 x 10(-6) M and > 10(-3) M were obtained with lyso-C16 and -C12, respectively. A structure model for a lipid-protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid-protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189-199).

Amino Acid Sequence↗
PubMed1995

Solution structure of a homopyrimidine: homopurine dodecamer encoded by the HIV-1 envelope gene: NMR and molecular simulation studies.

The solution structure of the nonpalindromic dodecanucleotide homopyrimidine:homopurine, d(5'-TTTCTCCTTTCT):d(5'-AGAAAGGAGAAA), was determined by two-dimensional nuclear magnetic resonance spectroscopy combined with molecular simulation. The dodecamer sequence studied was found within the HIV-1 envelope sequence and had all four Gs substituted by A in two hypermutants. A set of low-energy B-DNA conformations satisfying the quantitative NOE data were obtained. These highly related structure had neither peculiar helical parameters for the base pairs nor axis curvature. Analysis of the dihedral angles (epsilon-zeta) suggests that the A stretches flanking the GpA dinucleotides were more flexible.

Base Sequence↗
PubMed1995

Secondary structure in solution of the hydrophobic protein of soybean (HPS) as revealed by 1H NMR.

COSY, TOCSY and NOESY experiments have been used to assign sequentially the 1H 500 MHz NMR spectra of the Hydrophobic Protein of Soybean (HPS). Spin systems identification combined with sequential assignment allowed to identify the proton resonances of this 80 residues protein. Analysis of medium range connectivities showed that its secondary structure involved four helical fragments similarly located as in the structure deduced from X-ray diffraction. This work set the basis for a further fine comparison between the crystal and the solution structures and a dynamical study of HPS in solution. In addition, search of secondary structure similarities showed that the global folding of HPS should be rather similar to that found for non specific Lipid Transfer Proteins (ns-LTP) from vegetal origin. Distributions of the helical fragments along the primary sequences of these two classes of proteins were compared.

Amino Acid Sequence↗
PubMed1994

Two-dimensional 1H-NMR studies of maize lipid-transfer protein. Sequence-specific assignment and secondary structure.

Correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and NOE spectroscopy (NOESY) experiments have been used to assign sequentially the 1H 500-MHz NMR spectra of a non-specific (ns) lipid-transfer protein extracted from maize seeds. The spin-system identification and sequential assignment were combined with secondary-structure determination to identify most of the proton resonances of this 93-residue protein. From the sequential connectivities it was established that the secondary structure mainly involved four helical fragments: H1, H2, H3 and H4. This secondary structure was compared with that of wheat ns-lipid-transfer protein recently determined. The four helices are located in nearly the same regions, but helix H4 is appreciably longer in the maize protein than in the wheat protein. Comparison of the transfer activities reveals that the maize protein is more efficient than the wheat ns-lipid-transfer protein and that this difference is probably due to the affinity of the lipid for the binding site and not to the interfacial activation, i.e. adsorption of the ns-lipid-transfer protein to the membrane. From these results, it is suggested that helix H4 is a part of the lipid-binding site or contributes to the folding of this site. The present data define the basis for a further modelling of the three-dimensional structure of the maize ns-lipid-transfer protein which will be compared with that of the wheat ns-lipid-transfer protein in order to establish structure/activity relationships for this class of carriers by using natural ns-lipid-transfer protein mutants.

Amino Acid Sequence↗