Effect of pH on the structure and aggregation of human glycodelin A. A comparison with beta-lactoglobulin A

The effect of pH on the structure of glycodelin A (GdA) and of beta-lactoglobulin A (beta-LgA) has been investigated by means of circular dichroism, steady state fluorescence, synchrotron radiation small angle X-ray scattering (SR-SAXS) and gel permeation chromatography. The comparison between GdA and beta-LgA shows that, at pH 7.0, both proteins are dimers with an extended content of beta-sheet conformation, but pH 2.0 and 9.0 yield a different secondary, tertiary and quaternary structural organisation. Whilst beta-LgA is a monomer, that conserves beta-sheet conformation at pH 2.0 and 9.0, GdA has a stable dimeric structure at alkaline pH, but at pH 2.0 increases its alpha-helix content and it aggregates soon. SR beam has been used to perform SAXS comparative measurements of the two proteins. SR-SAXS data provide the radius of gyration and the radii of the cross-section and of the thickness. GdA aggregation at acid pH has been characterised by calculating the distance distribution function (P(r)). Isoelectric focusing and chromatofocusing data show a different charge distribution on the surfaces of the two proteins, supporting the hypothesis that the presence of oligosaccharides deeply influences the conformational state and the aggregation process of GdA at different pH values. In particular, the presence of sialic acid residues, within the oligosaccharide moiety of the GdA, might be responsible for the differences observed between the two proteins.     

Fluoroalcohols induced unfolding of Succinylated Con A: native like beta-structure in partially folded intermediate and alpha-helix in molten globule like state

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.     

Small-angle X-ray scattering study by synchrotron orbital radiation reveals that high molecular weight subunit of glutenin is a very anisotropic molecule.

Small-angle X-ray scattering of one high molecular weight (HMW) subunit of wheat glutenin was measured at protein concentration ranges from 1.0 to 10.0 mg/ml. The radius of gyration of whole particles, RO, in aq. 50% (v/v) 1-propanol and 0.1M acetic acid was 16.6 +/- 0.1nm and 22.8nm, respectively, and the corresponding radius of gyration of the cross-section, RC, was 2.82 +/- 0.02 nm and 2.23 +/- 0.01 nm, which indicate that the glutenin HMW subunit exists as very anisotropic particles in both solutions. The RO and RC values of the subunit, and the drastic decrease in scattered intensity at small angles that occurs in the acetic acid solution with relatively low protein concentration are completely explained in terms of rod-like molecules of the glutenin HMW subunit.     

Determination of the spatial structure of insectotoxin 15A from Buthus erpeus by (1)H-NMR spectroscopy data]

The solution structure of insectotoxin 15A (35 residues) from scorpion Buthus eupeus was determined on the basis of 386 interproton distance restraints 12 hydrogen-bonding restraints and 113 dihedral angle restraints derived from 1H NMR experiments. A group of 20 structures was calculated with the distance geometry program DIANA followed by the restrained energy minimization with the program CHARMM. The atomic RMS distribution about the mean coordinate position is 0.64 +/- 0.11 A for the backbone atoms and 1.35 +/- 0.20 A for all atoms. The structure contains an alpha-helix (residues 10-20) and a three-stranded antiparallel beta-sheet (residues 2-5, 24-28 and 29-33). A pairing of the eight cysteine residues of insectotoxin 15A was established basing on NMR data. Three disulfide bridges (residues 2-19, 16-31 and 20-33) connect the alpha-helix with the beta-sheet, and the fourth one (5-26) joins beta-strands together. The spatial fold of secondary structure elements (the alpha-helix and the beta-sheet) of the insectotoxin 15A is very similar to those of the other short and long scorpion toxins in spite of a low (about 20%) sequence homology.     

Structure and solvation of melittin in 1,1,1,3,3,3-hexafluoro-2-propanol/water

Fluorinated alcohols can induce peptides and proteins to take up helical conformations. Nuclear Overhauser effect (NOE) spectroscopy experiments and analysis of C(alpha)H proton chemical shifts show that the conformation of melittin in 35% hexafluoro-2-propanol/water is alpha-helical from residues Ile-2 to Val-8 and from Leu-13 to Gln-25. As has been found in other solvent systems, the two helical regions are not colinear; the interhelix angle (73 +/- 15 degrees ) in 35% 1,1,1,3,3,3-hexafluoro-2-propanol/water is smaller than the angle found in other fluoroalcohol-water mixtures or in the crystal. Intermolecular (1)H(19)F and (1)H(1)H nuclear Overhauser effects were used to explore interaction of solvent components with melittin dissolved in this solvent mixture. The NOEs observed indicate that fluoroalcohol and water molecules are both tightly bound to the peptide in the vicinity of the interhelix bend. For the remainder of the molecule, solute-solvent NOEs are consistent with preferential solvation of the peptide by the fluoroalcohol component of the solvent mixture.     

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.     

Structural studies of adenovirus type 2 by neutron and X-ray scattering

Small-angle neutron and X-ray scattering have been used to investigate various aspects of the structural organization of adenovirus type 2. Neutron scattering allows the determination of the radial distribution of DNA and protein, which because of the highly icosahedral form of the virus allows it to be described in terms of three icosahedral shells. X-ray scattering shows that the distance between the major coat proteins (hexons) in the capsid is 100 +/- A. Evidence was also observed for an organization in the nucleoprotein core that gives rise to a maximum in the X-ray scattering at 1/29 A-1.     

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.     

Determination of the secondary structure content of proteins in aqueous solutions from their amide I and amide II infrared bands. Comparison between classical and partial least-squares methods

A method for estimating protein secondary structure from infrared spectra has been developed. The infrared spectra of H2O solutions of 13 proteins of known crystal structure have been recorded and corrected for the spectral contribution of water in the amide I and II region by using the algorithm of Dousseau et al. [Dousseau, F., Therrien, M., & Pézolet, M. (1989) Appl. Spectrosc. 43, 538-542]. This calibration set of proteins has been analyzed by using either a classical least-squares (CLS) method or the partial least-squares (PLS) method. The pure-structure spectra calculated by the classical least-squares method are in good agreement with spectra of poly(L-lysine) in the alpha-helix, beta-sheet, and undefined conformations. The results show that the best agreement between the secondary structure determined by X-ray crystallography and that predicted by infrared spectroscopy is obtained when both the amide I and II bands are used to generate the calibration set, when the PLS method is used, and when it is assumed that the secondary structure of proteins is composed of only four types of structure: ordered and disordered alpha-helices, beta-sheet, and undefined conformation. Attempts to include turns in the secondary structure estimation have led to a loss of accuracy. The standard deviation of the difference between X-ray and infrared secondary structure estimates with this method is 4.8% for the alpha-helix, 3.7% for the beta-sheet, and 5.1% for the undefined structure, whereas the regression coefficients are 0.95, 0.96, and 0.56, respectively. The spectra of the calibration proteins were also recorded in 2H2O solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Fourier transform infrared spectroscopic (FTIR) study of porcine and bovine pancreatic phospholipase A2 and their interaction with substrate analogues and a transition-state inhibitor

Fourier transform infrared spectroscopy has been used to investigate the secondary structure of porcine and bovine pancreatic phospholipase A2 (PLA2) and the zymogen of porcine PLA2, prophospholipase A2 (proPLA2), in both H2O and D2O media. Detailed qualitative analysis was made of these proteins using second derivative and deconvolution techniques. Quantitative studies of the proteins in solution made using Factor Analysis gave average values of 54% alpha-helix, 15% beta-sheet and 23% beta-turns. These values agree well with the secondary structures deduced from previous studies of single crystals using X-ray techniques. No significant differences in secondary structure were observed for porcine pancreatic (pro)phospholipase A2 in the presence or absence of Ca2+ ions, or in the temperature range 10-45 degrees C. The binding of the non-degradable phospholipid analogue, n-alkylphosphocholine, in monomeric form produced no significant difference in the secondary structure of either enzyme. Conformational differences were, however, observed between the enzyme lyophilised in a solid film and in aqueous solution. The change is probably due to the formation of beta-sheet upon hydration, coupled with a loss of random structures. Conformational differences in both porcine and bovine pancreatic PLA2 were observed on binding to n-alkylphosphocholine micelles. This change may be due to a small increase in the alpha-helical structure and a decrease in the beta-sheet, and/or possibly beta-turn content. Similar conformational changes were observed for the interaction of porcine and bovine PLA2 with the substrate analogue inhibitor 1-heptanoyl-2-heptanoylamino-2-deoxy-sn-glycero-3-phospho glycol in micellar form.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues 20-83) of barley serine proteinase inhibitor 2 (BSPI-2) is determined on the basis of 403 interproton distance, 34 phi backbone torsion angle and 26 hydrogen bonding restraints derived from n.m.r. measurements. A total of 11 converged structures were computed using a metric matrix distance geometry algorithm and refined by restrained molecular dynamics. The average rms difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.2 A for the backbone atoms and 2.1 +/- 0.1 A for all atoms. The overall structure, which is almost identical to that found by X-ray crystallography, is disc shaped and consists of a central four component mixed parallel and antiparallel beta-sheet flanked by a 13 residue alpha-helix on one side and the reactive site loop on the other.     

Small angle X-ray scattering study on bovine porphobilinogen synthase (5-aminolaevulinate dehydratase)

The quaternary structure of the native (zinc) porphobilinogen synthase (5-amino-laevulinate dehydratase) from bovine liver and its lead-substituted derivative is studied in solution by small angle X-ray scattering. In spite of the profound inhibitory effect of lead ions in the enzyme they do not produce a change in the quaternary structure detectable by small angle X-ray scattering. The most important molecular parameters of the native enzyme were found to be: radius of gyration Rg = 4.04 +/- 0.04 nm and maximum dimension Dmax = 12.0 +/- 0.5 nm. The corresponding values for the lead derivative are: Rg = 4.26 +/- 0.1 nm and Dmax = 12.5 +/- 0.5 nm. The quaternary structure of the enzyme in solution is described by a model, which fits the experimental scattering and distance distribution function.     

Denaturation and aggregation of hen egg lysozyme in aqueous ethanol solution studied by dynamic light scattering

We applied dynamic light scattering technique on the model system of hen egg lysozyme in salt-free aqueous ethanol solution to study the mechanism of denaturation and aggregation of protein. At low ethanol concentration [0-63% (v/v)], the fast relaxation mode was observed, which was caused by lysozyme molecules in the solution interacting with each other with strong repulsive electrostatic force. At 45 and 63% (v/v) ethanol, the slow relaxation mode was also observed, which showed translational diffusive nature, similar to that observed in salt-free polyelectrolyte solution. At 72 or 81% (v/v) ethanol, the slow mode disappeared, leaving only the fast mode. However, the mutual diffusion coefficients obtained from the fast mode at 72 and 81% (v/v) ethanol decreased by about one order of magnitude compared with those from the fast mode at 0-63% (v/v). The reported alcohol-induced conformational transformation of lysozyme molecules at >60% (v/v) ethanol from their native structure to an alpha-helix-rich structure might cause such drastic decrease in the mutual diffusion coefficients. At the highest ethanol concentration of 90% (v/v), the slow mode reappeared, and its relaxation rate was decreasing with elapsed time, which is possibly due to the growth of aggregates of lysozyme molecules. X-ray diffraction results suggested that the intermolecular beta-sheet formation caused the aggregation. Thus, our results indicated that the change in molecular structure of lysozyme closely relates to the diffusion of molecules and their aggregation.     

Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences

Amino acid sequences, carbohydrate compositions and residue volumes are used to compare critically calculations of partial specific volumes v, neutron scattering matchpoints and 280-nm absorption coefficients with experimental v values for proteins and glycoproteins. The v values that are obtained from amino acid densitometry underestimate experimental v values by 0.01-0.02 ml/g while the v values from crystallographic volumes overestimate the experimental v values by 0.04-0.05 ml/g. An intermediate consensus volume set of amino-acid-residue volumes is proposed in order to predict experimental v values using sequence information. The method is extended to carbohydrates and glycoproteins. Neutron scattering matchpoints can be calculated from crystallographic residue volumes on the basis of the non-exchange of 10% of the main-chain NH protons. Crystallographic results on protein-bound water are used to account for the experimental values of v and matchpoints. Finally, 280-nm absorption coefficients, A1%, 1 cm 280, of 5-27 are found to be well predicted by the Wetlaufer procedure based on the totals of Trp, Tyr and Cys residues. Average errors are +/- 0.7, and the experimental A(1%,1cm)280 values can be larger than the predicted values by 3%.     

Conformation of human IgG subclasses in solution. Small-angle X-ray scattering and hydrodynamic studies

The structure of six human myeloma proteins: IgG1(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small-angle X-ray scattering and hydrodynamic methods. For IgG1(Bal) and IgG3(Het) the experimental data, including radius of gyration (Rg degree), radii of gyration of the cross-section (Rq1, Rq2), intrinsic viscosity [eta], sedimentation coefficient (S degree 20,w) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances. The values Rg degree = (6.00 +/- 0.05) nm, S degree 20,w = (6.81 +/- 0.10) S and [eta] = 0.0062 +/- 0.0005 cm3/mg obtained for IgG1(Bal) are compatible with a planar model in which the angle between the Fab arms is about 120 degrees. For IgG3(Het) the following data were obtained: Rg degree = (4.90 +/- 0.05) nm, S degree 20,w = (6.32 +/- 0.01) S and [eta] = (0.0065 +/- 0.0005) cm3/mg. The apparent contradiction between the higher molecular mass and lower Rg degree and S degree 20,w values for IgG3(Het) in comparison to IgG1(Bal) can be resolved by proposing a 'non-planar' (tetrahedral) molecular shape, in which the long hinge peptide is in a folded conformation and the two Fab and Fc parts are in a closely packed arrangement. In this model the angle between the two Fab arms is about 90 degrees, in the average position. The X-ray scattering and hydrodynamic behaviour of the IgG2 and IgG4 types of antibodies appeared to be similar to IgG1(Bal). The parameters of the two IgG3 proteins are similar while they are different to the others.     

Secondary structural analysis of the Na(+),K(+)-ATPase and its Na(+) (E(1)) and K(+) (E(2)) complexes by FTIR spectroscopy

The Na(+),K(+)-ATPase is an integral membrane protein which transports sodium and potassium cations against an electrochemical gradient. The transport of Na(+) and K(+) ions is presumably connected to an oscillation of the enzyme between the two conformational states, the E(1) (Na(+)) and the E(2) (K(+)) conformations. The E(1) and E(2) states have different affinities for ligand interaction. However, the determination of the secondary structure of this enzyme in its sodium and potassium forms has been the subject of much controversy. This study was designed to provide a quantitative analysis of the secondary structure of the Na(+),K(+)-ATPase in its sodium (E(1)) and potassium (E(2)) states in both H(2)O and D(2)O solutions at physiological pH, using Fourier transform infrared (FTIR) with its self-deconvolution and second derivative resolution enhancement methods, as well as curve-fitting procedures. Spectroscopic analysis showed that the secondary structure of the sodium salt of the Na(+),K(+)-ATPase in H(2)O solution contains alpha-helix 19.8+/-1%, beta-sheet 25.6+/-1%, turn 9.1+/-1%, and beta-anti 7.5+/-1%, whereas in D(2)O solution, the enzyme shows alpha-helix 16.8+/-1%, beta-sheet 24.5+/-1.5%, turn 10.9+/-1%, beta-anti 9.8+/-1%, and random coil 38.0+/-2%. Similarly, the potassium salt in H(2)O solution contains alpha-helix 16.6+/-1%, beta-sheet 26.4+/-1.5%, turn 8.9+/-1%, and beta-anti 8.1+/-1%, while in D(2)O solution it shows alpha-helix 16.2+/-1%, beta-sheet 24.5+/-1.5%, turn 10.3+/-1%, beta-anti 9.0+/-1%, and random coil 40+/-2%. Thus the main differences for the sodium and potassium forms of the Na(+),K(+)-ATPase are alpha-helix 3.2% in H(2)O and 0.6% in D(2)O, beta-sheet (pleated and anti) 1.5% in H(2)O and random structure 2% (D(2)O), while for other minor components (turn structure), the differences are less than 1%.     

E. coli trp repressor forms a domain-swapped array in aqueous alcohol

The E. coli trp repressor (trpR) homodimer recognizes its palindromic DNA binding site through a pair of flexible helix-turn-helix (HTH) motifs displayed on an intertwined helical core. Flexible N-terminal arms mediate association between dimers bound to tandem DNA sites. The 2.5 A X-ray structure of trpR crystallized in 30% (v/v) isopropanol reveals a substantial conformational rearrangement of HTH motifs and N-terminal arms, with the protein appearing in the unusual form of an ordered 3D domain-swapped supramolecular array. Small angle X-ray scattering measurements show that the self-association properties of trpR in solution are fundamentally altered by isopropanol.     

Synthetic protein design: construction of a four-stranded beta-sheet structure and evaluation of its integrity in methanol-water systems.

The characterization of a four-stranded beta-sheet structure in a designed 26-residue peptide Beta-4 is described. The sequence of Beta-4 (Arg-Gly-Thr-Ile-Lys-(D)pro-Gly-Ile-Thr-Phe-Ala-(D)Pro-Ala-Thr-Val-Leu-P he-Ala-Val-(D)Pro-Gly-Lys-Thr-Leu-Tyr-Arg) was chosen such that three strategically positioned (D)Pro-Xxx segments nucleate type II' beta-turns, which facilitate hairpin extension. A four-stranded beta-sheet structure is determined in methanol from 500 MHz 1H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal JCalphaH-NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four-stranded sheet and a nonpolar cluster involving Ile8, Phe10, Val15 and Phe17. Circular dichroism studies in water-methanol mixtures provide evidence for melting of the beta-sheet structure at high water concentrations. NMR analysis establishes that the four-stranded sheet in Beta-4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three beta-turn nuclei appear to be maintained.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: absence of the conserved structural water.

To probe the role of the Asp-99 ... His-48 pair in phospholipase A2 (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99-->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.