Characterization of two new crystal forms of uteroglobin.

Two new crystal forms of oxidized uteroglobin have been obtained. An orthorhombic one ($\text{P2}{}_1\text{2}{}_1\text{2, Z = 2, a = 44.48 (5)}\text{A}\text{?}\text{, b = 36.93 (5)}\text{A}\text{?}\text{, c = 32·34 (5)}\text{A}\text{?}$) and a monoclinic one ($\text{P2}{}_1\text{, Z = 2, a = 44.56 (5)}\text{A}\text{?}\text{, b = 46.06 (5)}\text{A}\text{?}\text{, c = 37.43 (4)}\text{A}\text{?}\text{, β = 120.92 ° (5)}$). Both were grown at pH ~7.0 and diffract to a resolution of 2·1 to 2·2 Å. Data collections for native crystals have been recorded with an automatic four-circle diffractometer.     

Cytoskeletal proteins used as marker of differentiation in mouse terato;carcinoma cells

The cyclic AMP receptor protein (CRP) of Escherichia coli has been crystallized. The crystals are orthorhombic, space group $\text{P2}{}_1\text{2}{}_1\text{2}{}_1\text{, a = 46.5}\text{A}\text{?}$, b = 97.1 Å, $\text{c = 105.4}\text{A}\text{?}$, with one dimeric CRP molecule per asymmetric unit.     

Preliminary x-ray crystallographic data for the semisynthetic non-covalent complex of residues 1 to 118 and 111 to 124 of bovine pancreatic ribonuclease

The enzymically active, semisynthetic complex formed by residues 1 through 118 and residues 111 through 124 of bovine pancreatic ribonuclease has been crystallized at pH 5.7 from $\text{(}\text{NH}{}_4\text{)}{}_2\text{SO}{}_4\text{CsCl}$ solutions. The crystals belong to space group P3₂21, have unit cell dimensions $\text{a}\text{and}\text{b = 64.4}\text{A}\text{?}\text{, c = 64.5}\text{A}\text{?}\text{,}\text{and}$ γ = 120° and are isomorphous with form M of ribonuclease A as well as forms W and R of ribonuclease S. They diffract well and may be expected to yield a structure defined to at least 3.0 Å resolution.     

Crystallization of alfalfa mosaic virus coat protein as a T = 1 aggregate

Reassembled alfalfa mosaic virus coat protein was partially digested with trypsin to remove the first 26 amino acids (Bol et al., 1974). These particles are empty icosahedral protein shells built with 60 alfalfa mosaic virus protein subunits. This aggregate has been crystallized in two different crystal forms, one of which diffracts X-rays to at least 3.4 Å resolution. The type I crystals (space group $\text{P6}{}_3\text{, a = 200}\text{A}\text{?}\text{, c = 314}\text{A}\text{?}$) contain two particles per cell separated by 195 Å with each sitting on a 3-fold axis. The type II crystals contain three particles per cell in space group P3₁or P3₂ ($\text{a = 201}\text{A}\text{?}\text{, c = 485}\text{A}\text{?}$). Other T = 1 viral particles have very similar diameters.     

Yeast transfer RNAasp: a new high-resolution x-ray diffracting crystal form of a transfer RNA.

Systematic crystallization studies on brewer's yeast aspartic acid transfer RNA have yielded different crystal forms, one of them diffracting to 3 Å resolution. The high resolution crystal form is orthorhombic ($\text{C222}{}_1\text{, a = 61}\text{A}\text{?}\text{, b = 68}\text{A}\text{?}\text{, c = 148}\text{A}\text{?}$ with one molecule per asymmetric unit) and is stable for over four days under X-rays.     

Electron diffraction study on single crystals of l-type and dl-type lecithins

An electron diffraction study was carried out on thin single micro-crystals of l-type and dl-type dipalmitoyl lecithins grown in xylene suspensions and fine net patterns were obtained and the mechanism of the thermotropic phase transitions of them was clarified.From the apparent structure of diffraction patterns in low temperature, it is confirmed that the two dimensional lattices have p mm symmetry in l-type and in dl-type lecithins. Lattice parameters from the [001] projection are $\text{d}{}_{100}\text{= 9.9}\text{A}\text{?}$ and $\text{d}{}_{010}\text{= 8.8}\text{A}\text{?}$ in l-type, and $\text{d}{}_{100}\text{= 17.2}\text{A}\text{?}$ and $\text{d}{}_{010}\text{= 8.9}\text{A}\text{?}$ in dl-type.With anisotropic variation of dimensions along a and b axes, i.e. contraction for a and expansion for b, induced by temperature rise by electron irradiation during the observation, these diffraction patterns of the lattices of l-type and dl-type were transformed into those characterized by the six diffraction spots having nearly the same spacings. Four of them are observed on slightly outer and two are slightly inner positions as compared with their mean spacings of about (4.1 Å)^?1 in l-type and about (4.2 Å)^?1 in dl-type. The changes in the patterns observed indicate that at low temperatures the hydrocarbon chains are nearly perpendicular to the layer in dl-type lipid, and tilted with a more complicated packing in l-type ones. The dimension along a in dl-type is twice as large as that in l-type.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Crystallographic studies of immunoglobulins: crystallization of the Fc fragment of rabbit IgG with and without cleavage of the inter-chain disulphide bridge

The Fc fragment was prepared from rabbit immunoglobulin G by digestion with papain, both with the inter-chain disulphide bond intact, and after reduction and alkylation. These two types of Fc crystallized in different, yet related forms, each with one dimer in the asymmetric unit. The covalently linked dimer crystallized in space group $\text{P2}{}_1\text{; a = 68.85 ± 0.05}\text{A}\text{?}\text{, b = 72.50 ± 0.05}\text{A}\text{?}\text{, c = 60.40 ± 0.05}\text{A}\text{?}$ and β = 105.1 ± 0.2 °. The reduced, non-covalently linked dimer also crystallized in space group $\text{P2}{}_1\text{; a = 81.55 ± 0.05}\text{A}\text{?}\text{, b = 55.65 ± 0.05}\text{A}\text{?}\text{, c = 68.85 ± 0.05}\text{A}\text{?}$ and β = 1051 ± 0.2 °. A non-crystallographic 2-fold axis relating the two identical polypeptide chains is clearly visible in the h0l projection of the second crystal form.     

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes. Determination of the equilibrium between QA−QB and QAQB−

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q_A^?Q_B and Q_AQ_B^?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of $\text{α =}\text{[}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{]}\text{([}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{] + [}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}{}^{\mathrm{?}}\text{])}\text{= 0.065 ± 0.005}$ (T = 21°C, pH 8).     

Structure of the semiquinone form of flavodoxin from Clostridum MP. Extension of 1.8 A resolution and some comparisons with the oxidized state

As part of a series of comparisons of the structures of the three oxidation states of flavodoxin from Clostridium MP, phases for the semiquinono form were determined to 2.0 Å resolution by isomorphous replacement (〈m 〉 = 0.725). Subsequently, the structure was refined at 1.8 Å resolution by a combination of difference Fourier, real space and reciprocal space methods. After refining to an R of 0.194, we explored the conformation of the FMN binding site by real space refinement versus maps with Fourier coefficients of the form ($\text{2¦F}{}_{\mathrm{<mtext>o</mtext>}}\text{¦? ¦F}{}_{\mathrm{<mtext>c</mtext>}}\text{¦)}\text{exp}\text{(iα}{}_{\mathrm{<mtext>c</mtext>}}$). To minimize bias in the fitting, groups of atoms were systematically omitted from the structure factors used in computation of the ($\text{2¦F}{}_{\mathrm{<mtext>o</mtext>}}\text{? ¦F}{}_{\mathrm{<mtext>c</mtext>}}\text{¦}$) maps.One-electron reduction of oxidized flavodoxin is accompanied by several changes at the FMN binding site: the conformation of residues in the reverse bend formed by Met56-Gly57-Asp58-Glu59 differs in the crystal structures of the oxidized and semiquinone species; further, backbone atoms in residues 55 and 89 shift by more than 0.5 Å and the indole ring of Trp90 undergoes a significant displacement. The orientation of the peptide unit connecting Gly57 and Asp58 is consistent with the presence of a hydrogen bond between the carbonyl oxygen of Gly57 and the flavin N(5) in flavodoxin semiquinone. No equivalent bond is found in oxidized flavodoxin. In both the oxidized and semiquinone species of clostridial flavodoxin, the isoalloxazine ring is essentially planar : the bending angles about N(5)N(10) are ~2.5 ° for the semiquinone structure and ~0 ° in oxidized flavodoxin.The intensity changes resulting from the oxidized agsemiquinone conversion (R_I = 0.33) arise in part from changes in molecular packing. Intermolecular contacts, including neighbors of the prosthetic group, are altered in the repacking. Maps or models of the two oxidation states can be brought into approximate coincidence by a rigid body motion. The required transformation, determined for the isomorphous replacement maps by the method of Cox (1967), is equivalent to a screw motion with a rotation of 1.18 ° and a translation of ?0.34 Å. The molecular structures of oxidized and semiquinone flavodoxins have been compared after superposition of models with idealized co-ordinates and discrepancy indices Rox = 0.213 and Rsq = 0.200. The root-mean-square distance between 523 backbone atoms (excluding sequences 56 to 59 and 89 to 91) is 0.308 Å.     

Crystallographic data for a penicillin receptor : exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61.

A pencillin-sensitive enzyme, the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (M_r = 6000 to 7500) solution at pH 7·6. X-ray examination of the orthorhombic crystals shows the space group is P2₁2₁2₁, with unit cell dimensions $\text{a = 51·1}\text{A}\text{?}\text{, b = 67·4}\text{A}\text{?}\text{,}\text{and}\text{c = 102·9}\text{A}\text{?}$. With four molecules of molecular weight 38,000, the $\text{A}\text{?}{}^3\text{/}\text{dalton}$ ratio for the cell is 2·33. The crystals are stable to irradiation for 75 hours and are suitable for structure analysis to at least 2·4 Å resolution. The radius of gyration of the molecule in solution at pH 6.8 is 20.8 Å.     

Photophosphorylation in bacterial chromatophores entrapped in alginate gel: Improvement of the physical and biochemical properties of gel beads with barium as gel-inducing agent

The immobilization of Rhodopseudomonas capsulata chromatophores by entrapment in an alginate gel is described. Alginate beads were prepared with Ba²⁺, Sr²⁺ and Ca²⁺ as gel-forming agents and compared for their mechanical strength, chemical resistance against disruption by phosphate-induced swelling, and yield of photophosphorylation activity. Barium alginate beads proved to have better physico-chemical properties than the more commonly used calcium alginate beads. After embedding in barium alginate gel, R. capsulata chromatophores retained a high yield (up to 70%) of their photophosphorylation capacity. Alginate entrapment did not cause a large increase in the Michaelis constant for ADP and phosphate, the substrates of adenosinetriphosphatase (ATPase). These constants were $\text{K}{}^{\mathrm{ADP}}{}_{\mathrm{<mtext>m</mtext>}}\text{= 1.4 × 10}{}^{\mathrm{?5}}\text{m}$ and $\text{K}{}^{\mathrm{P}}{}_{\mathrm{i}}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.2 × 10}{}^{\mathrm{?4}}\text{m}$ for free chromatophores and $\text{K}{}^{\mathrm{ADP}}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.3 × 10}{}^{\mathrm{?4}}\text{m}$ and $\text{K}{}^{\mathrm{P}}{}_{\mathrm{i}}{}_{\mathrm{<mtext>m</mtext>}}\text{= 5.6 × 10}{}^{\mathrm{?4}}\text{m}$ for chromatophores entrapped in barium alginate gel. However, embedding gave no additional protection against rapid inactivation of chromatophores upon storage at 3°C. Preliminary results with a batch reactor for continuous ATP regeneration are presented. The barium alginate method has two features which are not generally encountered at the same time, extremely mild conditions for entrapment and excellent physical properties of the gels beads, which make this method a suitable tool for the construction of bioreactors with immobilized cells or organelles.     

Preliminary crystallographic data for actinidin,a thiol protease from Actinidia chinensis

Crystals of actinidin, a thiol protease from the fruit of Actinidia chinensis, which are suitable for high-resolution X-ray diffraction studies, have been obtained. The space-group is $\text{P2}{}_1\text{2}{}_1\text{2}{}_1\text{,}\text{with}\text{a = 78.1}\text{A}\text{?}\text{, 6 = 81.2}\text{A}\text{?}\text{and}\text{c = 33.0}\text{A}\text{?}$. The asymmetric unit contains one molecule, of molecular weight about 26,000.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.     

A preliminary crystallographic study of wheat germ agglutinin

Wheat germ agglutinin crystallizes in two monoclinic space groups, P2₁ and C2, under identical crystallization conditions. Unit cell dimensions are $\text{a = 73.8}\text{A}\text{?}\text{, b = 51.2}\text{A}\text{?}\text{, c = 90.8}\text{A}\text{?}\text{, γ = 90 °}$ for $\text{P2}{}_1\text{; a = 51.31}\text{A}\text{?}\text{, b = 73.35}\text{A}\text{?}\text{, c = 91.45}\text{A}\text{?}\text{, β = 97.75 °}$ for C2, both with eight subunit molecules in the unit cell. The C2 crystals were chosen as suitable for investigating the three-dimensional structure to high resolution, because of their smaller asymmetric unit (containing the dimer), and also because they display better diffraction patterns.     

Characterization of a monoclinic crystal form of an enzyme of steroid metabolism, Δ5-3-ketosteroid isomerase

A monoclinic crystal form ($\text{P2}{}_1\text{, a = 140.4}\text{A}\text{?}\text{, b = 85.0}\text{A}\text{?}\text{, c = 94.5}\text{A}\text{?}$, β= 130.1 °) of Δ⁵-3-ketosteroid isomerase from Pseudomonas testosteroni (EC 5.3.3.1), grown at pH 7.0, has been characterized. Crystal-density measurements show that the asymmetric unit contains 12 protomers (M_r = 13,394).     

Evidence for general catalysis and formation of nitrobenzene in the oxidation of phenylhydroxylamine in aqueous phosphate buffer

N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O₂ dependent and shows general catalysis by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}$ (k₁ = 2.3 M^?2 sec^?1) and $\text{PO}{}_4{}^{\mathrm{?3}}$ (k₂ = 2.3 × 10⁵M^?2 sec^?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.     

Laser-light scattering investigation of the micellar properties of gangliosides

The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. G_M1 and G_D1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c₀) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius R_H, and also contained information on the polydispersity of the sample. We find c_o < 1 × 10^?6 M for both G_M1 and G_D1a, M = 532 000 ± 50 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 63.9 ± 2}\text{A}\text{?}$ for G_M1, and M = 417 000 ± 40 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 59.5 ± 2}\text{A}\text{?}$ for G_D1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca²⁺, did not influence significantly the values of c_o and the main features of the micelle.     

Crystallization of resolvase, a repressor that also catalyzes site-specific DNA recombination

Resolvase, a site-specific recombination enzyme involved in transposition of movable elements of DNA, has been crystallized. The space group is P6₂22 (or enantiomorph $\text{P6}{}_4\text{22, a = b = 59.7}\text{A}\text{?}\text{, c = 169.4}\text{A}\text{?}$), with a monomer in the crystallographic asymmetric unit.