Finding protein-protein interaction patterns by contact map matching

We propose a novel method for defining patterns of contacts present in protein-protein complexes. A new use of the traditional contact maps (more frequently used for representation of the intra-chain contacts) is presented for analysis of inter-chain contacts. Using an algorithm based on image processing techniques, we can compare protein-protein interaction maps and also obtain a dissimilarity score between them. The same algorithm used to compare the maps can align the contacts of all the complexes and be helpful in the determination of a pattern of conserved interactions at the interfaces. We present an example for the application of this method by analyzing the pattern of interaction of bovine pancreatic trypsin inhibitors and trypsins, chymotrypsins, a thrombin, a matriptase, and a kallikrein - all classified as serine proteases. We found 20 contacts conserved in trypsins and chymotrypsins and 3 specific ones are present in all the serine protease complexes studied. The method was able to identify important contacts for the protein family studied and the results are in agreement with the literature.

On the characterization of energy networks of proteins

The construction of a realistic theoretical model of proteins is determinant for improving the computational simulations of their structural and functional aspects. Modeling proteins as a network of non-covalent connections between the atoms of amino acid residues has shown valuable insights into these macromolecules. The energy-related properties of protein structures are known to be very important in molecular dynamics. However, these same properties have been neglected when the protein structures are modeled as networks of atoms and amino acid residues. A new approach for the construction of protein models based on a network of atoms is presented. This method, based on interatomic interaction, takes into account the energy and geometric aspects of the protein structures that were not employed before, such as atomic occlusion inside the protein, the use of solvation, protein modeling and analysis, and the use of energy potentials to estimate the energies of interatomic non-covalent contacts. As a result, we achieved a more realistic network model of proteins. This model has the virtue of being more robust in face of different unknown variables that usually are arbitrarily estimated. We were able to determine the most connected residues of all the proteins studied, so that we are now in a better condition to study their structural role.

A contact map matching approach to protein structure similarity analysis

We modeled the problem of identifying how close two proteins are structurally by measuring the dissimilarity of their contact maps. These contact maps are colored images, in which the chromatic information encodes the chemical nature of the contacts. We studied two conceptually distinct image-processing algorithms to measure the dissimilarity between these contact maps; one was a content-based image retrieval method, and the other was based on image registration. In experiments with contact maps constructed from the protein data bank, our approach was able to identify, with greater than 80% precision, instances of monomers of apolipoproteins, globins, plastocyanins, retinol binding proteins and thioredoxins, among the monomers of Protein Data Bank Select. The image registration approach was only slightly more accurate than the content-based image retrieval approach.

The Star STING server: a multiplatform environment for protein structure analysis

Star STING is the latest version of the STING suite of programs and corresponding database. We report on five important aspects of this package that have acquired some new characteristics, designed to add key advantages to the whole suite: 1) availability for most popular platforms and browsers, 2) introduction of the STING_DB quality assessment, 3) improvement in algorithms for calculation of three STING parameters, 4) introduction of five new STING modules, and 5) expansion of the existing modules. Star STING is freely accessible at: http://sms.cbi.cnptia.embrapa.br/SMS/, http://trantor.bioc.columbia.edu/SMS, http://www.es.embnet.org/SMS/, http://gibk26.bse.kyutech.ac.jp/SMS/ and http://www.ar.embnet.org/SMS.

Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine ß-trypsin (homogeneous material, observed 23,294.4 ± 0.2 Da, theoretical 23,292.5 Da) with the inhibitors benzamidine and berenil at pH 8.0, 25°C and with 25 mM CaCl2, were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 ± 25,057 and 49,513 ± 2,732 M-1, respectively; the number of binding sites is the same for both ligands, N = 0.99 ± 0.05). Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (DCp), a characteristic signature of these interactions, was similar in both systems tested (-464.7 ± 23.9 and -477.1 ± 86.8 J K-1 mol-1 for berenil and benzamidine, respectively). The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of ß-trypsin may result in more effective inhibitors.

Characterization of á-trypsin at acid pH by differential scanning calorimetry

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel á-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of á-trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM á-alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (deltaHcal/deltaHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, deltaHcal/deltaHvH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, á-trypsin unfolded with Tm = 54ºC and deltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (deltaCp) was estimated to be 2.50 ± 0.07 kcal mol-1 K-1. The stability of á-trypsin calculated at 298 K was deltaG D = 5.7 kcal/mol at pH 3.00 and deltaG D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media

Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 `M) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 `M), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 `M) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 + or - 0.8 `M and k cat = 48.4 + or - 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 + or - 10, 1,098 + or - 91, 38.6 + or - 5.2 and 37,340 + or - 5,400 `M, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 + or - 92.8 and 310,500 + or - 38,600 `M, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits 3H-choline uptake in guinea pig ileum

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of 3H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 ÁM) and up to 10 min of treatment, crotoxin decreased 3H-choline uptake by 50-75 percent compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 ÁM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on 3H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits 3H-choline uptake with high affinity (EC25 = 10 +/- 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission

Partially folded intermediates during trypsinogen denaturation

The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was delta GH2O = 6.99 + ou - 1.40 kcal/mol for guanidine hydrochloride and delta GH2O = 6.37 + ou - 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 + ou - 0.4 angstron to 26.0 + ou - 0.3 angstron 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 + ou - 0.3 angstron to 25.7 + ou - 0.6 angstron for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine ß-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using a-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of ß-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 µM; kinetic parameters for the substrate hydrolysis were: Km = 32 µM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme

Ph titration of native and unfolded beta-trypsin: evaluation of the delta delta G§ titration and the carboxyl pK values

The stabilizing free energy of beta-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (delta delta G° titration) was 9.51 + 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in beta-trypsin. In fact, one class of carboxyl group with a pK= 3.91 + 0.01 and another one with a pK= 4.63 + 0.03 were also found by hydrogen in titration of the protein in the folded state.

Kinetic characterization of rat tissue kallikrein using NÓ-substituted arginine 4-nitroanilides and NÓ-benzoyl-L-arginine ethyl ester as substrates

Hydrolysis of seven N(alpha-substituted L-arginine 4-nitroanilides: henzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N(alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 muM for p-nitroanilides and 50 to 190 muM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 muM for p-nitroanilides and 200 to 1800 muM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 ñ 2 muM; Vmax 10.42 ñ 0.28 muM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 ñ 2 muM; Vmax = 3.21 ñ 0.11 muM/min), while Tos-Arg-Nan (Km = 29 ñ 2 muM; Vmax, = 0. 10 ñ 0.002 muM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 ñ 15 muM; Vmax = 121.3 ñ 7.6 muM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.

Kinetic mechanism of the inhibition of human urinary kallikrein by basic pancreatic trypsin inhibitor

Hydrolysis of D-valvyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentration (10-20µM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15nM). The data indicate that the inhbition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formetion of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, repectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule

Partial purification of an endoglucanase from Biomphalaria glabrata

Previous studies have shown that Biomphalaria glabrata contains a complete cellulolytic system which includes an endoglucanase, an exoglucanase and a ß-glucosidase. In the present report, a scheme for the purification of the endoglucanase from this invertebrate is proposed. Two major problems were encountered during the study: 1) the presence of a green-brownish pigment, which could not be eliminated by thermal shock or ammonium sulfate precipitation and 2) relative instability of enzymatic activity. Various alternatives were tested and the best sequence of steps was: 1) a sample of the crude extract, obtained by homogenization of the digestive glands in 50 mM Tris-HCl buffer, pH 8.4, and ultracentrifugation, was applied to a Q-Sepharose FPLC column (50 mM Tris-HCl buffer, pH 8.4; 10 mm x 22.2 cm column; flow rate 1.5 ml/min; 0.1 to 0.5 M NaCl gradient); 2) the eluate peak containing activity was dialyzed, lyophilized and eluted from a Superdex-75 gel filtration FPLC column (50 mM ammonium acetate buffer, pH 4.8; 16 mm x 60 cm column; flow rate 1.0 ml/min). A low degree of purification (about 36-fold) and recovery (about 12 percent) were observed, probably due to enzyme instability. SDS-electrophoresis of the active fraction showed a major peak of 30 kDa. In order to improve the purification scheme, further studies are required to stabilize this enzyme during purification and storage