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A Tulinsky

Publications and source records attributed to A Tulinsky.

At least 91 records · Page 5Linked to original sources
PubMed1988

Preliminary x-ray investigation of an orthorhombic crystal of hevein.

Hevein, a small protein from the latex of Hevea brasiliensis, has been crystallized by the vapor diffusion method using 2-methyl-2,4-pentanediol and CaCl2 as the precipitant agents. The crystals are orthorhombic space group P21212 with a = 21.88, b = 31.90, and c = 51.24 A and one molecule in the asymmetric unit. The crystals are quite stable to x-rays and suitable for a high resolution three-dimensional structure determination.

Antimicrobial Cationic Peptides↗
PubMed1988

Lysine/fibrin binding sites of kringles modeled after the structure of kringle 1 of prothrombin.

The Lys binding site of kringle 1 and 4 (K1 and K4) of plasminogen (PG) has been modeled on the basis of the three-dimensional structure of kringle 1 of prothrombin and 300- and 600-MHZ proton nuclear magnetic resonance observations. These structures were then compared to the corresponding regions of modeled kringle 1 and 2 of tissue plasminogen activator (PA). The coordinates of the modeled structures have been refined by energy minimization in the presence and absence of epsilon-aminocaproic acid ligand in order basically to remove unacceptable van der Waals contacts. The binding site is characterized by an apparent dipolar surface, the polar parts of which are separated by a hydrophobic region of highly conserved aromatic residues. Zwitterionic ligands such as Lys and epsilon-aminocaproic acid form ion pair interactions with Asp55 and Asp57 located on the dipolar surface; the latter are also conserved in all the Lys binding kringles. The cationic center of the dipolar surface is Arg71, in the case of PGK4, and is composed of Arg34 and Arg71 in PGK1. The doubly charged anionic/cationic interaction centers of the latter might account for the larger binding constants of PGK1 for like-ligands but the modeling suggests that PGK4 might be kinetically faster in binding bulkier ligands. The binding site region of PAK2, which also binds Lys, resembles those of PGK1 and PGK4. Since PAK2 lacks both cationic center Arg residues, ligand carboxylate binding appears to be accomplished though an imidazolium ion of His64, which is located just below the outer surface of the kringle.

Amino Acid Sequence↗
PubMed1987

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site.

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.

Binding Sites↗
PubMed1987

Complete assignment of the aromatic proton magnetic resonance spectrum of the kringle 1 domain from human plasminogen: structure of the ligand-binding site.

The kringle 1 domain of human plasminogen has been investigated by 1H NMR spectroscopy at 300 and 600 MHz on the basis of a fragment obtained via controlled proteolysis of the zymogen with Staphylococcus aureus V8 protease. The aromatic spectrum has been fully analyzed and all resonances assigned. The Tyr ring signals were identified by reference to the recently reported spectra of the plasminogen kringle 4 homologues from human, bovine, and porcine origin [Ramesh, V., Gyenes, M., Patthy, L., & Llinás, M. (1986) Eur. J. Biochem. 159, 581-595]. In particular, Tyr74 was assigned on the basis of a proton Overhauser experiment showing cross-relaxation with the Trp-II (Trp62) indole ring, a connectivity previously observed in all the kringle 4 variants and that clearly represents a conserved feature of the kringle structure. Ligand binding was investigated by monitoring the effects of the antifibrinolytic drugs epsilon-aminohexanoic acid and p-benzylaminesulfonic acid (BASA) on the 1H NMR spectrum of kringle 1. It is observed that although most aromatic resonances are perturbed by ligand presence, the chemical shift response is significantly more marked for Phe36, Trp62, and Tyr72. Proton Overhauser experiments centered on aromatic transitions from these residues reveal efficient cross-relaxation with BASA, which indicates direct contacts between the hydrophobic side chain rings and the ligand hydrocarbon moiety at the binding site. A close interaction is also found between Tyr64 and Try72 which indicates that the residue 64 ring is positioned close to the binding site. Excellent overall agreement is found between the NMR data and the molecular folding of the prothrombin kringle 1 determined crystallographically [Park, C. H., & Tulinsky, A. (1986) Biochemistry 25, 3977-3982]. A structure is proposed here for the kringle 1 lysine-binding site which is based upon the NMR results, the X-ray structure, and computer graphics modeling. It is concluded that although features of the lysine-binding site are common to plasminogen kringles 1 and 4, in kringle 1 the binding site extends beyond the kringle inner loop as it encompasses residues Arg34 and Phe36 as well. Furthermore, it appears that in kringle 1 Arg34 and Asp55 are likely to play a direct role in the ligand-kringle 1 interaction by reinforcing the polarity of the cationic and anionic centers of the side chains of Arg71 and Asp57, which have been implicated to provide the electrostatic charges in kringle 4 that balance those of the ligand dipole at the binding site.

Amino Acid Sequence↗
PubMed1987

Structure of a tetrahedral transition state complex of alpha-chymotrypsin dimer at 1.8-A resolution.

A 1.8-A resolution x-ray crystallographic restrained least squares refinement has been carried out on the phenylethane boronic acid (PEBA) complex of alpha-chymotrypsin dimer (alpha-CHT), and it has been compared to the 1.67-A resolution structure of the native enzyme. PEBA has a high binding affinity for alpha-CHT, and the boronate forms a tetrahedral complex with Ser-195 OG of one molecule of the dimer; the boronate in the other molecule is severely disordered and does not form a tetrahedral complex. The former could be a model of the transition state of catalysis. The complex of PEBA X alpha-CHT displays significant nonequivalence in conformation of side chains between the independent molecules comparable to the native enzyme, but, like the latter, shows a high degree of fidelity in the folding of the main chain. The orientation of the phenyl ring, CA and CB of PEBA, in the specificity sites of the two molecules is similar, suggesting that recognition is fairly insensitive to small departures from local symmetry; the same does not apply to the boronate functionalities suggesting that greater precision is required for catalysis. The folding of the molecule remains the same upon PEBA binding, but some of the side chains respond nonequivalently. The latter is a consequence of the inherent nonequivalence of the native dimer and the asymmetrical nature of the PEBA binding.

Binding Sites↗
PubMed1986

Three-dimensional structure of the kringle sequence: structure of prothrombin fragment 1.

The three-dimensional structure of bovine prothrombin fragment 1 has been solved at 2.8-A resolution. The electron density clearly reveals four disulfide bridges along with more than 80% of the side chains completely in density, which correspond faithfully to the kringle sequence, its preceding 30 residues, and the dodecapeptide carboxy terminal; the polysaccharide and the first 35 residues of the amino terminal of fragment 1 are disordered or about 40% of the structure. The folding of the kringle sequence is based upon close disulfide van der Waals contacts between Cys-87-Cys-127 and Cys-115-Cys-139 (4.1 A between midpoints of the bridges), two antiparallel strands of highly conserved (113-118, 124-129) beta-structure, and the stacking of some conserved aromatic residues, all near the center of the folded structure. Moreover, the overall folding appears to be duplicated as a pair of stacked duplex loops with an antiparallel open loop. The overall shape of the kringle structure approximates an eccentric oblate ellipsoid of dimensions 11 X 28 X 30 A. The residues immediately preceding the kringle are dominated by alpha-helical structure (Phe-41-Cys-48; Leu-56-Glu-63). Residues Phe-41-Trp-42 and Tyr-45, which are conserved in factor IX, factor X, protein C, and protein Z, form another aromatic stacked cluster while the Cys-48-Cys-61 disulfide loop corresponds to the well-known alpha/beta structural unit. The dodecapeptide carboxy-terminal interkringle chain extends along the periphery of the kringle in its plane and forms a beta-structure with the kringle-closing Ser-140-Val-143 tetrapeptide.

Amino Acid Sequence↗
PubMed1986

Preliminary crystallographic data and quaternary structural implications of the central subunit of the multi-subunit complex transcarboxylase.

The hexameric central subunit (Mr = 360,000) of the multi-subunit complex transcarboxylase has been crystallized by bulk dialysis against 250 mM-sodium acetate (pH 5.5). The crystals are cubic, a = 193.1 A, space group P4(1)32 or enantiomorph. The number of molecules per unit cell is four and was deduced from the density of the crystals (1.10 g cm-3) and the mother liquor (1.01 g cm-3) and the specific volume of the protein calculated from molecular dimensions obtained from electron microscopy studies. Four molecules per cell requires the central subunits to lie on 3-fold axes, which are perpendicular to 2-fold rotation axes, so that the molecules satisfy 32 symmetry giving one subunit as the asymmetric unit. Of the four possible models that have been considered for the quaternary structure of transcarboxylase, only that with antiparallel subunits, two sets of isologous binding sites and D3 symmetry is in agreement with the symmetry requirements of the cubic crystals.

Carboxyl and Carbamoyl Transferases↗
PubMed1985

The structure of prothrombin fragment 1 at 3.5-A resolution.

The structure of prothrombin fragment 1 has been determined at 3.5-A resolution by multiple isomorphous replacement methods with four heavy atom derivatives. The final average figure of merit is 0.72. There is a large cylindrical solvent region with an average diameter of 35-40 A along the entire length of the c axis (85 A) centered at about x = y = 1/2. The connected density forming the wall of this channel is not of sufficient extent to account for the 156 residues of fragment 1 and the two accompanying carbohydrate chains totaling 5000 in molecular weight. Deglycosylated fragment 1 crystallizes isomorphously with fragment 1, and a difference map between the two revealed that the sugar chains are severely disordered and reside in the solvent channel. Although the disordered carbohydrate and the complexity of five disulfides in a 126-residue sequence have hampered the complete tracing of the peptide chain, two-thirds of the molecule has been accounted for in the form of an unusually oblate ellipsoid of about 15 X 30 X 35 A. The folding of the molecule has little secondary structure (one alpha-helix (7%), 20% beta-structure) in agreement with dichroism measurements and one of the points of carbohydrate attachment is suggested from the deglycosylated difference map.

Amino Acid Sequence↗
PubMed1985

Comparison of the independent solvent structures of dimeric alpha-chymotrypsin with themselves and with gamma-chymotrypsin.

The solvent structure of alpha-chymotrypsin has been determined in the restrained least squares refinement (1.67-A resolution) of the dimeric molecule (Blevins, R. A., and Tulinsky, A. (1985) J. Biol. Chem. 260, 4264-4275). A total of 247 water molecules reduced the R-factor by 0.039 to 0.179. The average occupancy of solvent is 0.77 and the average isotropic thermal parameter is 22 A2. About 80% of the solvent is around the surface, 10% is in the dimer interface, and 10% is interior. There are 49 pairs of water molecules related by 2-fold noncrystallographic symmetry (within 1.0 A) and 199 waters that can potentially hydrogen bond with protein or themselves. The specificity sites contain 5 water molecules, 2 of which are displaced by substrate binding. The remainder probably aid in identifying and positioning the latter for catalysis. Four of these waters also occur in gamma-chymotrypsin. Considering the water structure in the dimer interface region of alpha-chymotrypsin with that of gamma-chymotrypsin reveals that about two-thirds of the solvent in this region is lost on dimerization. Last, 4 of the water molecules of alpha-chymotrypsin have been identified to be sulfate ions from a difference map based on crystals with selenate exchanged mother liquor.

Binding Sites↗
PubMed1985

The refinement and the structure of the dimer of alpha-chymotrypsin at 1.67-A resolution.

The two molecules of the asymmetric unit of the pH 3.5 conformer of alpha-chymotrypsin have been refined at 1.67-A resolution using restrained least squares methods with Hendrickson's program (PROLSQ). The final R factor is 0.179 (including 247 water molecules). The folding of the main chain of the independent molecules is the same within experimental error but the same does not generally apply to the side chain stereochemistry. From this we conclude that the folding of a protein structure is basically independent of most of the detailed stereochemistry of its side chains. The side chains of the interface region between the independent molecules display pronounced asymmetry. This asymmetry suggests that dynamic and asymmetrical structural changes take place at the time of oligomerization leading to more energetically favorable interactions for the dimer. Comparison of the structures of the independent molecules of alpha-chymotrypsin with the structure of monomeric gamma-chymotrypsin revealed that although the folding of the three molecules is essentially the same, numerous and significant differences pervade the side chain stereochemistry attributable to general flexibility. The specificity site of alpha-chymotrypsin is occupied by ordered water molecules in a similar way to gamma-chymotrypsin and other proteins. Some of these water molecules are displaced when substrate binds to the enzyme, while the others appear to help identify and position the aromatic side chain in catalysis.

Binding Sites↗
PubMed1981

Formation, crystallization, and preliminary crystallographic data of the ternary complex of alpha-chymotrypsin, beta-trypsin, and the Bowman-Birk inhibitor.

The ternary complex of the Bowman-Birk inhibitor with alpha-chymotrypsin and beta-trypsin has been crystallized and preliminary crystallographic information describing the crystals has been obtained. The crystals are triclinic with unit cell dimensions: a = 51.96 A, b = 56.34 A, c = 46.70 A, alpha = 98.83 degrees, beta = 97.29 degrees, and gamma = 97.15 degrees. There is 1 ternary complex/unit cell and the diffraction pattern extends to 2.2 A resolution. The search for heavy atom derivatives based on known derivatives of chymotrypsin and trypsin is underway.

Chymotrypsin↗
PubMed1980

Crystallization and preliminary X-ray data of proteins derived from prothrombin.

Crystals of bovine prothrombin fragment 1 and deglycosylated fragment 1 have been grown from polyethylene glycol, Tris/maleate solutions and the crystals scatter x-rays at 2.8 A resolution. The crystals of both are tetragonal, space group P41212 or P43212, with 1 molecule per asymmetric unit. Moreover, the two appear to be isomorphous. Since the mother liquor can support Ca2+ ion without precipitation, crystals have been soaked with Ca2+ ion and preliminary results indicate that these remain closely isomorphous with parent crystals. Isomorphous heavy atom derivatives of mercury and platinum have been prepared of glycosylated fragment 1. In addition, crystals of human prothrombin fragment 1 have also been grown.

Animals↗
PubMed1979

Interaction of alpha-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution.

The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.

Allosteric Site↗