Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4.

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays major roles in fertilization and consists of three or four glycoproteins. Primary structures, and especially the positions of cysteine (Cys) residues in the zona glycoproteins, are well conserved among mammals. In this study, we analyzed the disulfide linkages of pig ZP3 and ZP4 purified from ovaries. While disulfide linkage patterns of four Cys residues in the N-terminal halves of the ZP domains of ZP3 and ZP4 were identical to those previously reported for mice, rats, humans, and fish, the disulfide linkage patterns of six Cys residues in the C-terminal half of the ZP domain in ZP4, as well as eight Cys residues in the C-terminal region of the ZP domain and a following region unique to ZP3, were different from those previously reported. Thus, higher-order structures of zona glycoproteins might not be conserved in the C-terminal regions.

The three-dimensional structure of septum site-determining protein MinD from Pyrococcus horikoshii OT3 in complex with Mg-ADP.

BACKGROUND: In Escherichia coli, the cell division site is determined by the cooperative activity of min operon products MinC, MinD, and MinE. MinC is a nonspecific inhibitor of the septum protein FtsZ, and MinE is the supressor of MinC. MinD plays a multifunctional role. It is a membrane-associated ATPase and is a septum site-determining factor through the activation and regulation of MinC and MinE. MinD is also known to undergo a rapid pole-to-pole oscillation movement in vivo as observed by fluorescent microscopy. RESULTS: The three-dimensional structure of the MinD-2 from Pyrococcus horikoshii OT3 (PH0612) has been determined at 2.3 A resolution by X-ray crystallography using the Se-Met MAD method. The molecule consists of a beta sheet with 7 parallel and 1 antiparallel strands and 11 peripheral alpha helices. It contains the classical mononucleotide binding loop with bound ADP and magnesium ion, which is consistent with the suggested ATPase activity. CONCLUSIONS: Structure analysis shows that MinD is most similar to nitrogenase iron protein, which is a member of the P loop-containing nucleotide triphosphate hydrolase superfamily of proteins. Unlike nitrogenase or other member proteins that normally work as a dimer, MinD was present as a monomer in the crystal. Both the 31P NMR and Malachite Green method exhibited relatively low levels of ATPase activity. These facts suggest that MinD may work as a molecular switch in the multiprotein complex in bacterial cell division.

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides.

The 1H-NMR titration curves of chemical shifts versus pH were observed for imidazole, N1-methylimidazole, L-histidine, N1-methyl-L-histidine, N3-methyl-L-histidine, and other related compounds. With these results, the macroscopic pK values of these compounds were determined by a computer curve-fitting for a simple dissociation sequence. From the pK values of imidazole and N1-methylimidazole, the perturbation for the pK of the imidazole ring due to the substitution of a proton with a methyl group was estimated as -0.21 pH unit. The microscopic pK values of the individual tautomers of the imidazole ring were estimated with the pK values of N1-methyl-L-histidine, N3-methyl-L-histidine, and perturbation due to methyl substitution. The estimated pK values were 6.73 for the N1-H tautomer and 6.12 for the N3-H tautomer. These values were in good agreement with those obtained using carboxymethyl derivatives instead of methyl derivatives. Furthermore, the macroscopic pK value (6.02) calculated using the estimated microscopic pK values agreed with that (6.03) observed for the imidazole ring of L-histidine. Thus the method in this work was indicated to be self-consistent. The microscopic pK values of tautomers were also obtained for N alpha-acetyl-L-histidine and N alpha-acetyl-L-histidine methylamide. The molar ratios of tautomers were calculated on the basis of the microscopic pK values of tautomers. The intrinsic (or unperturbed) pK value of imidazole ring and perturbations due to the CO2- and NH3+ were obtained for each of the N1-H and N3-H tautomers.

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. II. Microenvironments of histidine-12 and histidine-119 of bovine pancreatic ribonuclease A.

The NMR titration curves of proton chemical shifts were observed for the C2 protons of histidine residues in intact bovine pancreatic RNAase A (EC 3.1.27.5) and carboxyalkylated RNAase A. By comparing the methyl region of NMR spectra, the 250-340 nm region of circular dichoic spectra, and the NMR titration curves of tyrosine ring protons among intact and modified RNAase A, it was ascertained that the carboxyalkylation of histidine residues at position 12 or 119 did not make any appreciable conformational changes to RNAase A. With the pK values determined for intact and modified RNAase A, the microscopic pK values and molar ratios of tautomers were estimated for His-12 and His-119 by means of the procedure described in the preceding paper. The estimated microscopic pK values of tautomers were 6.2 for the N1-H tautomer of His-12, more than 8 for the N3-H tautomer of His-12, 7.0 for the N1-H tautomer of His-119, and 6.4 for the N3-H tautomer of His-119, respectively. These values were interpreted in terms of the microscopic environments surrounding the histidine residues. The microscopic structure estimated in the present study was discussed, comparing it with those from X-ray crystallography and hydrogen-tritium (or hydrogen-deuterium) exchange technique.

Crystallization and preliminary X-ray investigation of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus.

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase distinctly different in various properties from the family of pepsin-type aspartic proteinases, and so far it remains unknown which residues participate in the catalysis of the enzyme and how the mechanism operates. The acid proteinase A was crystallized from an ammonium sulfate solution by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1) with unit cell dimensions of a = 54.7 A, b = 70.4 A and c = 38.0 A. On the assumption that there is one enzyme molecule in the asymmetric unit, the calculated ratio of volume to unit protein mass (Vm) was 1.64 A3 per dalton. Diffraction data were collected up to a resolution higher than 1.5 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. The crystal of proteinase A is, therefore, suitable for the structural analysis with a high resolution.

1.8 A crystal structure of the major NAD(P)H:FMN oxidoreductase of a bioluminescent bacterium, Vibrio fischeri: overall structure, cofactor and substrate-analog binding, and comparison with related flavoproteins.

We have solved the crystal structure of FRase I, the major NAD(P)H:FMN oxidoreductase of Vibrio fischeri, by the multiple isomorphous replacement method (MIR) at 1.8 A resolution with the conventional R factor of 0.187. The crystal structure of FRase I complexed with its competitive inhibitor, dicoumarol, has also been solved at 2.2 A resolution with the conventional R factor of 0.161. FRase I is a homodimer, having one FMN cofactor per subunit, which is situated at the interface of two subunits. The overall fold can be divided into two domains; 80% of the residues form a rigid core and the remaining, a small flexible domain. The overall core folding is similar to those of an NADPH-dependent flavin reductase of Vibrio harveyi (FRP) and the NADH oxidase of Thermus thermophilus (NOX) in spite of the very low identity in amino acid sequences (10% with FRP and 21% with NOX). 56% of alpha-carbons of FRase I core residues could be superposed onto NOX counterparts with an r.m.s. distance of 1.2 A. The remaining residues have relatively high B-values and may be essential for defining the substrate specificity. Indeed, one of them, Phe124, was found to participate in the binding of dicoumarol through stacking to one of the rings of dicoumarol. Upon binding of dicoumarol, most of the exposed re-face of the FMN cofactor is buried, which is consistent with the ping pong bi bi catalytic mechanism.

Changes in intracellular pH and inorganic phosphate concentration during and after muscle contraction as studied by time-resolved 31P-NMR. Alkalinization by contraction.

The changes in intracellular pH and concentration of inorganic phosphate (Pi) were studied during and after contraction of bullfrog sartorius muscles by using 31P-NMR. The intracellular pH was 7.04 in resting muscles at 4 degrees C and an alkalinization by as much as 0.08 pH unit occurred following 5 s tetanic contraction. During the recovery period after contraction the increased level of intracellular pH returned to the resting level, the time course of which coincided with that of the recovery of the Pi concentration. Based on the changes in the intracellular pH and Pi concentration, the buffering power was estimated to be 18 mM/pH unit.

A calorimetric study of Ca2+ binding to two major isotypes of bullfrog parvalbumin.

Microcalorimetric titrations of the two major isotypes of parvalbumin (PA1 and PA2) from bullfrog skeletal muscle with Ca2+ in the presence and absence of Mg2+ have been carried out at 25 degrees C and pH 7.0. The observed enthalpy titration curves were analyzed by the least-squares method. The measured enthalpy changes (delta H) of Ca2+ binding are -33.2 (PA1) and -16.3 kJ/mol site (PA2), and the entropy changes (delta S) are 28 (PA1) and 76 j/mol per K (PA2) in the absence of Mg2+. When 5 mM Mg2+ is present, the enthalpy change of PA2 (-26.7 kJ/mol) is about twice as large as that in the absence of Mg2+, whereas that of PA1 (-34.6 kJ/mol) is about the same. The entropy changes are 8 (PA1) and 29 J/mol per K (PA2). Both enthalpy and entropy changes are favorable for the Ca2+-binding reactions of PA1 and PA2 irrespective of the presence of Mg2+.

The complete amino acid sequence of bullfrog (Rana catesbeiana) parvalbumin pI4.97.

The primary structure of the parvalbumin (pI4.97) from bullfrog, Rana catesbeiana, skeletal muscle has been determined. It is composed of 110 amino acid residues and a free amino terminus, and has a molecular mass of 11919. The amino acid sequences which are thought to be functionally important sites are also conserved in the bullfrog parvalbumin. The calculated phylogenic tree indicates that this parvalbumin belongs to the alpha group of parvalbumins. The mutation rate of parvalbumin was fairly rapid in frogs compared to mammals. The subdivergence of frogs is also discussed.

A calorimetric study of Ca2+ binding by the parvalbumin of the toad (Bufo): distinguishable binding sites in the molecule.

Microcalorimetric titrations of the major isotype of parvalbumin (tPA) from toad (Bufo) skeletal muscle, with Ca2+ in the presence and absence of Mg2+ and with Mg2+ in the absence of Ca2+, have been carried out at 25 degrees C and pH 7.0. The results indicate that the two binding sites in each molecule are distinguishable from each other for both Ca2+ binding and Mg2+ binding. Such a characteristic is distinctly different from those of other parvalbumins. The enthalpy changes determined are distinctly different from those of bullfrog parvalbumins on Ca2+ or Mg2+ binding, but are similar to those on Mg2+-Ca2+ exchange. The results indicate that the reaction of Mg2+-Ca2+ exchange is driven almost entirely by the large favorable enthalpy change.

Hydrolysis of AMPPNP by the motor domain of ncd, a kinesin-related protein.

AMPPNP was found to be hydrolyzed by the motor domain of ncd (the product of a Drosophila gene, non-claret disjunctional), a kinesin-related protein. This hydrolysis could be monitored by 31P NMR spectroscopy and by an assay of phosphate, one of the products of the hydrolysis. The rate was approximately 0.00004 s(-1), 1% of the ATP turnover rate. The AMPPNP turnover was not stimulated by microtubules. Kinesin motor domain also turned over AMPPNP but at a somewhat lower rate. Although the turnover was slow, the present finding may present an important caveat, since AMPPNP has been widely used for investigations of kinesin and kinesin-related proteins as a non-hydrolyzable ATP analogue.

Infrared studies of interaction between metal ions and Ca(2+)-binding proteins. Marker bands for identifying the types of coordination of the side-chain COO- groups to metal ions in pike parvalbumin (pI = 4.10).

Metal-ligand interactions in the Ca(2+)-binding sites of pike parvalbumin (pI = 4.10) have been examined by Fourier-transform infrared spectroscopy. The region of the COO- antisymmetric stretch provides useful information on the types of coordination of the COO- groups to the metal ions in the Mg(2+)-, Mn(2+)-, and Ca(2+)-bound forms. In the spectrum of the Ca(2+)-bound form, two bands are observed at 1,582 and 1,553 cm-1, whereas, in the spectra of the Mg(2+)- and Mn(2+)-bound forms, bands are observed only in the region around 1,582 cm-1 and no band is found in the region around 1,553 cm-1. The 1,553-cm-1 band of the Ca(2+)-bound form reflects the bidentate coordination of the COO- groups of both Glu-62 in the CD site and Glu-101 in the EF site to the Ca2+ ions, which has been made clear by X-ray analysis as a feature of the Ca(2+)-bound form. Absence of such a band in the spectrum of the Mn(2+)-bound form is consistent with the X-ray structure of this form where both of the two COO- groups are unidentate. These unidentate COO- groups of Glu-62 and Glu-101 in the Mn(2+)-bound form seem to give rise to a band at 1,577-1,574 cm-1. The spectrum of the Mg(2+)-bound form is also consistent with the 'pseudo-bridging' coordination of the COO- group of Glu-101 reported in the X-ray structure of a form where the Mg2+ ion occupies only the EF site, and the same spectrum is further indicative of the 'pseudo-bridging' coordination of the COO- group of Glu-62.

Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6.

Kinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Akaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR.

Thermal stabilization of ribonuclease T1 by carboxymethylation at Glu-58 as revealed by 1H nuclear magnetic resonance spectroscopy.

Ribonuclease T1 (RNase T1) carboxymethylated at the gamma-carboxyl group of Glu-58 with iodoacetic acid is known to be completely inactive while it retains an almost full substrate-binding ability. In order to further clarify the effects of the carboxymethylation, the thermal stabilities of intact and Glu-58-carboxymethylated (CM-) RNase T1 were compared by measuring 1H NMR spectra at various temperatures. The transition curves of unfolding were obtained by plotting, as a function of temperature, the peak areas for the alpha and delta protons of Asn-81 and Ile-90, respectively, which are well apart from each other in the three-dimensional structure of the enzyme. For each of intact and CM-RNase T1, the transition curve of the Asn-81 alpha proton was identical with that of the Ile-90 delta methyl protons, suggesting that the thermal unfolding occurred simultaneously in every part of the molecule of CM-RNase T1 as well as of intact RNase T1. The midpoint of unfolding was 52 degrees C for intact RNase T1, and was increased by 9 degrees C upon carboxymethylation at Glu-58. This marked stabilization by carboxymethylation is thought to be due to formation of a salt bridge between the introduced carboxymethyl group and the neighboring guanidium group of Arg-77.

1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A.

The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.

Interaction of substrate analogs with bovine pancreatic ribonuclease A as studied by 1H nuclear magnetic resonance.

The 270 MHz 1H NMR spectra of 3'-UMP and 3'-CMP were observed in the presence of a two-fold molar excess of bovine pancreatic RNase A [EC 3.1.27.5] at various pHs. For the C(5), C(6), and C(1') protons of these nucleotides, the pH profiles of chemical shifts induced by binding to RNase A were obtained by plotting the differences between chemical shifts in the presence and the absence of RNase A against pH. Such profiles were bell-shaped for the C(5) and C(6) protons of both 3'-UMP and 3'-CMP. However the profiles of C(1') protons were not bell-shaped but appeared to consist of two bell-shaped curves and reflect the dissociations of at least three ionizable groups. The observations for the C(1') protons suggest that there are at least two forms of complexes different from each other in the interaction reflecting the chemical shift of the C(1') proton. In order to clarify the interacting sites of ribonucleotides affecting the induced shift profile of the C(1') proton, the pH titration curves were observed for 3'-dCMP in the presence of RNase A. The induced shift profile was bell-shaped for the C(1') proton as well as for the C(5) proton of the base. This indicates that the interaction at the O(2')H [or O(2')] sites of ribonucleotides causes the two forms of complexes of 3'-UMP and 3'-CMP with RNase A. The interacting sites and modes were discussed with these and the pH titration curves of His-12, His-119, and Phe-120 of RNase A in the presence of a three-fold molar excess of ribonucleotides.

Steady-state properties of calcium binding to parvalbumins from bullfrog skeletal muscle: effects of Mg2+, pH, ionic strength, and temperature.

To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI 4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I = 0.106, pH 6.80, at 20 degrees C) containing various concentrations of Mg2+ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca2+ in the presence of Mg2+ changed in the manner expected if Ca2+ and Mg2+ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, KCa = 1 X 10(7) M-1, KMg = 900 M-1; for PA-2, KCa = 6 X 10(6) M-1, KMg = 830 M-1 (I = 0.106, pH 6.80, at 20 degrees C). The apparent binding constants are strongly dependent on temperature: at 10 degrees C for PA-1, KCa = 2 X 10(8) M-1, KMg = 10(4) M-1; for PA-2, KCa = 5 X 10(7) M-1, KMg = 5 X 10(3) M-1 (I = 0.106, pH 6.80). The dependence of the affinities for Ca2+ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca2+ and Mg2+ of parvalbumins are unchanged between pH 6.5 and 7.2.

Kinetic studies of calcium binding to parvalbumins from bullfrog skeletal muscle.

In addition to steady-state properties of calcium binding to parvalbumins, kinetic studies are required for adequate evaluation of the physiological roles of parvalbumins. By using a dual-wavelength spectrophotometer equipped with a stopped-flow accessory, the transient kinetics of calcium binding to parvalbumins (PA-1 and 2) from bullfrog skeletal muscle was examined at 20 degrees C in medium containing 20 mM MOPS-KOH, pH 6.80, 0.13 mM tetramethylmurexide, 25 microM CaCl2, metal-deprived PA-1 or PA-2, various concentrations of Mg2+, and KCl to adjust the ionic strength of the medium to 0.106. The results can be explained in terms of the following rate constants under the conditions mentioned above when a second-order kinetic scheme is assumed. For PA-1, the association and apparent dissociation rate constants for Ca2+ are 1.5 X 10(7) M-1 X s-1 and 1.5 s-1, respectively, or more. The rate constants for Mg2+ are 7,500 M-1 X s-1 and 5-6 s-1, respectively. For PA-2, the rate constants for Ca2+ are 7 X 10(6) M-1 X s-1 and 1.16 s-1, respectively, and those for Mg2+ are 3,500 M-1 X s-1 and 3.5-4 s-1, respectively. Increased affinities for Ca2+ and Mg2+ at 10 degrees C are largely due to decreased apparent dissociation rate constants for these divalent cations, because no significant change in the association rate constants was found.

Complexes of myosin subfragment 1 with pyrophosphate and with adenosine diphosphate as studied by phosphorus-31 nuclear magnetic resonance.

Myosin subfragment-1.pyrophosphate (S1.PPi) complex and S1.ADP complex were observed with 31P NMR at various temperatures between 0 and 25 degrees C. The signal of S1.PPi complex showed a small temperature dependence, indicating that a single conformation exists for the complex. It also showed that the electron density around phosphorus nuclei was increased upon the formation of complex. On the other hand, the signal of S1.ADP complex was clearly dependent on temperature and indicated the presence of two forms, i.e., high-temperature and low-temperature forms. In the high-temperature form, the electron density around beta-phosphate was decreased upon the formation of complex with S1. In the low-temperature form, the distribution of electrons around phosphorus nuclei is extremely anisotropic due to the tight interaction of S1 and the phosphate moieties of ADP.

A calorimetric study of Ca2+- and Mg2+-binding by calmodulin.

Enthalpy titrations of calmodulin with Ca2+ (or Mg2+) in the presence and the absence of Mg2+ (or Ca2+) have been carried out by microcalorimetry. The binding reactions of both Ca2+ and Mg2+ to calmodulin are endothermic, indicating that the large entropy change gives rise to the strong binding of Ca2+ to calmodulin. The results also indicated that all the four binding sites are Ca2+-Mg2+ sites. These results are markedly different from the findings for troponin C.