Small-angle X-ray scattering studies of Escherichia colil-asparaginase

Small angle X-ray scattering studies on Escherichia colil-asparaginase solutions show that the enzyme has a radius of gyration of 34.0 Å ± 0.5 Å at pH 7. The radius of gyration of the dissociated monomer is 16.0 Å ± 1.0 Å; it has the general shape of a prolate ellipsoid with an axial ratio of 1.4. A tetramer of four such ellipsoids arranged with 222 symmetry gives good agreement between measured and calculated radii of gyration if the distance between subunit centers is 43 Å. The tetramer dissociates on dilution below 1% and at pH values below 3.0. Acid-induced denaturation at pH 2.0 is irreversible in contrast to the reversible guanidine-HCl-induced denaturation.     

Distribution of bacteria in the velocity gradient centrifuge.

Cells in different parts of the cell cycle can be separated by brief centrifugation in a density stabilized gradient: the Mitchison-Vincent technique. The position of a cell in the tube depends upon its size, shape, and density, upon the gradients of density, viscosity, and centrifugal force through which it sediments, and upon time. A program to compute the velocities and integrate the velocity profile for particles of a particular size class is presented. Because enteric bacteria are a form intermediate between right cylinders and prolate ellipsoids of revolution, the program uses values for the frictional coefficient intermediate between those calculated for ellipsoids and for cylinders. The formula f=6pietab(a/b)1/2 possesses this property and because of its simplicity greatly speeds the calculations. A second program computes the distribution of masses and then of sedimentation constants for a bacterial population, expressed either as a frequency distribution or as total mass per s-class. The effect of the known variation in cell size at division is included in these calculations, which apply to organisms undergoing balanced, asynchronous growth in which mass increase is proportional to cell size. The two programs in conjunction compute the mass or cell-number profile in an arbitrary gradient. The programs have been used to design gradients to maximize the resolution of the technique.     

Identification and Characterization of a New Lipoprotein, NlpI, in Escherichia coli K-12

We report here the identification of a new lipoprotein, NlpI, in Escherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp (polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.     

The dipolar origin of protein relaxation

1. A set of parameters is proposed to check the interpretation of the dielectric behaviour of protein solutions as a rigid-dipole relaxation of prolate ellipsoids of revolution in the frequency range between 20 kHz and 10 MHz. Besides the delta(b)-function of Scheraga, another analogous function (delta(a)) is presented to establish size and shape of globular proteins. A study of the influence of solvent viscosity on the dielectric dispersion also gives strong evidence in favour of rigid-dipole relaxation. 2. Measurements of the dielectric dispersion of monomer solutions of bovine serum albumin and transferrin are reported. Monomers of bovine serum albumin were obtained by fractionation on Sephadex G-150. Low-conductivity solutions of both proteins are obtained by passage through an ion-exchange resin. 3. Computer analysis of the experimental dispersion curves by use of a two-term Debye dispersion gives valuable information about transferrin and leads to an axial ratio 4.5 for a prolate ellipsoid of revolution. The dielectric increment of bovine serum albumin is very low and no conclusive results have yet been obtained.     

SEDIMENTATION AND VISCOSITY STUDIES ON THE CAPSULAR AND SOMATIC POLYSACCHARIDES OF PNEUMOCOCCUS TYPE III

Sedimentation constants at infinite dilution have been found to be 1.89 and 4.06 for the somatic and capsular polysaccharides, respectively, from pneumococcus Type III. Intrinsic viscosities have been determined for the somatic and capsular polysaccharides of pneumococcus Type III using the Ostwald viscometer. Molecular weights and dimensions have been calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be prolate ellipsoids of revolution. Values for the somatic polysaccharide are: molecular weight, 26,400; diameter, 0.97 mµ; and length, 36.18 mµ. Values for the capsular polysaccharide are: molecular weight, 171,800; diameter, 1.04 mµ; and length, 177.87 mµ. The molecular weights were calculated for the somatic and capsular polysaccharides of pneumococcus Type III assuming the molecules to be flexible chains. The value of the molecular weight of the somatic polysaccharide is 31,500 and the value for the molecular weight of the capsular polysaccharide is 267,500. The molecules of both the somatic and capsular polysaccharides exhibit high degrees of asymmetry.     

Numerical inversion of the Perrin equations for rotational and translational diffusion constants by iterative techniques.

An iterative numerical technique is presented which allows the semiaxes for prolate and oblate ellipsoids to be determined from the Perrin equations for rotational and translational diffusion constants. The use of this inversion technique is illustrated by application to the proteins: lysozyme, bovine serum albumin, human transferrin, and bovine rhodopsin solubilized in digitonin.     

Shape of protein L11 from the 50S ribosomal subunit of Escherichia coli.

Protein L11 from the 50S ribosomal subunit of Escherichia coli A19 was purified by a method using nondenaturing conditions. Its shape in solution was studied by hydrodynamic and low-angle x-ray scattering experiments. The results from both methods are in good agreement. In buffers similar to the ribosomal reconstitution buffer, the protein is monomeric at concentrations up to 3 mg/mL and has a molecular weight of 16 000-17 000. The protein molecule resembles a prolate ellipsoid with an axial ratio of 5-6:1 a radius of gyration of 34 A, and a maximal length of 150 A. From the low-angle x-ray diffraction data, a more refined model of the protein molecule has been constructed consisting of two ellipsoids joined by their long axes.     

Dynamic light scattering from dilute suspensions of thin discs and thin rods as limiting forms of cylinder, ellipsoid and ellipsoidal shell of revolution

A previous formulation of the field correlation function G1(tau) of light quasielastically scattered from suspensions of rigid rods undergoing anisotropic translational as well as rotational diffusion (T. Maeda and S. Fujime, Macromolecules 17 (1984) 1157) was extended to the cases of suspensions of cylinders (length L and radius R), ellipsoids and ellipsoidal shells of revolution (x2/b2 + y2/b2 + z2/a2 = 1). The present formulation includes that for suspensions of rigid rods in the limit of KR 1 or in the limit of b/a 1 and Kb 1 (an extremely prolate ellipsoid), and also that for suspensions of discs in the limit of KL 1 or in the limit of b/a 1 and Ka 1 (an extremely oblate ellipsoid), where K is the length of the scattering vector. Explicit forms of G1(tau), of the first cumulant Gamma of G1(tau) and of the dynamic form factors will be given, and numerical methods suitable for computation of dynamic form factors will be discussed. The present results can be applied to the analysis of experimental data for dilute suspensions of thin rods and thin discs. When the situation is favorable, our method can provide transport coefficients D1, D3, and Theta from dynamic light-scattering data only, where D(1) and D(3) are, respectively, the translational diffusion coefficients parallel with the x (y) and z axes, and Theta the rotational diffusion coefficient around the x (y) axis.     

Frictional coefficients of multisubunit structures. I. Theory

The theory of Kirkwood for the translational frictional coefficients of structures composed of subunits has been generalized in two ways in order to consider aggregates of nonidentical subunits. One of these generalizations fails when the sizes of subunits are too disparate; the other, derived from a surface shell distribution of frictional elements, is effective over the whole range of relative sizes. It is shown that, in the limit of a continuous surface distribution, a shell model reproduces Stoke's law for a sphere. Comparison is made between the frictional coefficients of spheres, ellipsoids, and rods modeled by finite numbers of subunits and by continuous shells of frictional elements, and those calculated from other theories. Agreement is generally good, though the shell model for prolate ellipsoids of revolution deviates by a few per cent from the Perrin value.     

Fluorescence polarization studies of human and rhesus A-I apolipoproteins and their complexes with phosphatidylcholine.

Human and rhesus A-I apolipoproteins, covalently labelled with dansyl chloride, were used in fluorescence polarization studies of: 1) the monomeric structure of the free proteins in solution; 2) the interaction of the apolipoproteins with sonicated egg phosphatidylcholine; and 3) the size of the saturated complexes of protein with phospholipid. The results indicate that both monomeric apolipoproteins have relatively rigid, yet asymmetrical structures, with Stokes radii of 24.2 ± 0.5 Å, in neutral aqueous solutions. Axial ratios are of the order of $\text{6}\text{1}$ or $\text{4}\text{1}$ for hydrated, prolate or oblate ellipsoids, respectively. A molar excess of about 200 phosphatidylcholine molecules are required to saturate each apolipoprotein. At saturation, the complexes with both proteins have Stokes radii of 40.6 ± 1.7 Å. Since the radius of phosphatidylcholine vesicles is around 125 Å, we conclude that the complexes are relatively small structures derived from disruption of the lipid vesicles, rather than from adsorption of the proteins on intact vesicles.     

The combination of the viscosity increment with the harmonic mean rotational relaxation time for determining the conformation of biological macromolecules in solution.

A new hydrodynamic shape function, lambda, is derived for determining the conformation of biological macromolecules in solution, adding to the increasing number of shape parameters whose experimental determination does not require a knowledge of the particle swelling due to solvation in solution. lambda can be found from a knowledge of the molecular weight, intrinsic viscosity and the harmonic mean rotational relaxation time. A table of values and a plot of lambda as a function of axial ratio for both oblate and prolate ellipsoids of revolution are given.     

Translational friction of a particle inside a vesicle studied by an extended shell method

The translational friction of a particle inside a rigid hollow sphere is determined in this work. This should be important in model studies of solute diffusion in cells or vesicles. An extended shell algorithm is developed and used in which the cell or vesicle is modeled as a large number of beads. In the extended algorithm, the shell is divided into groups of beads and the assumption is made that the force exerted by each bead on the solvent is constant within a group. The algorithm is tested by using it to determine the translational friction constants of spheres and prolate ellipsoids modeled as shells of beads.     

Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin

Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations. The HMW subunit existed as a highly extended rod-like particle in solution with a length of about 69 nm and a diameter of about 6.4 nm. Specific aggregation effects were observed which may reflect mechanisms of self-assembly that contribute to the unique viscoelastic properties of wheat dough.     

Ultraviolet Optical Rotatory Dispersion of Aspergillopeptidase A

Some physicochemical properties of the acid-stable α-amylase and the acid-unstable α-amylase, produced by Asp. niger simultaneously, were investigated comparing with each other. The molecular weights of the acid-stable α-amylase and the acid-unstable α-amylase were computed from sedimentation equilibrium as 58,000 and 61,000, respectively. And the molecular shapes were investigated by measuring sedimentation coefficient and intrinsic viscosity. The results indicated that these two α-amylases were similar in hydrodynamic properties and that they were prolate ellipsoids and poor hydrated proteins. But high frictional ratio of the acid-stable α-amylase suggested a certain degree of molecular asymmetry. Isoelectric points of the acid-stable α-amylase and the acid-unstable α-amylase were determined as pH 3.44 and 3.70, respectively.     

A study of rhodopsin-detergent micelles by transient electric birefringence.

The hydrodynamic method of transient electric birefringence has been used to study bovine rhodopsin solubilized in two detergents, 0.02% Ammonyx LO and 0.045% digitonin. All measurements are interpreted as the sum of two exponentials by which the relaxation times yield the rotary diffusion coefficients for ellipsoids of revolution. The semi-major and minor axes for prolate ellipsoid models have been calculated and their axial ratio, 6.8, in both detergents, is in line with recent reports on the structure of rhodopsin. Studies on bleached rhodopsin showed a large increase in axial ratio in 0.02% Ammonyx LO.     

The molecular parameters of monomeric and acid-soluble collagens. Low shear gradient viscosity and electric birefringence

The molecular parameters of pronase-treated acid-soluble bovine skin collagen (P-ASC) were determined from low-shear gradient viscosity, electric birefringence, and electron microscopic data in order to determine the shear gradient range in which viscosity studies yield data which can be correctly interpreted by use of the various hydrodynamic equations for prolate ellipsoids of revolution. The P-ASC solutions could be characterized by a single relaxation process in electric briefringence with rotary diffusion coefficient θ of 810 sec^?1 and a corresponding molecular length of 2850 Å. Viscosity data were found to be shear gradient dependent and only the extrapolated zero-shear value [η]D = 0 could be used with the viscosity hydrodynamic equations to provide a correct value of molecular length. Intrinsic viscosities obtained at shear gradients >250 sec^?1 are nearly 30% lower than the zero-shear value. Untreated acid-soluble collagen (ASC) solutions contain aggregates and these appear, from electric birefringence data, to be of endlinked character. ASC solutions show a much more marked shear gradient dependence than P-ASC. For example, at D～500sec^?1,[η] = 22 dl/g, whereas the extrapolated zero-shear value of[η] was found to be 44 dl/g. Thus, the shear gradient dependence of native collagen solutions is much more marked than previously assumed and, in contrast to the usual practice, only viscosities measured near zero shear can be interpreted in terms of molecular parameters for collagen solutions containing aggregates.     

Affinity chromatography and properties of cGMP-dependent protein kinase from prawn tissues

Using affinity chromatography on 8-(2-aminoethyl)-amino-cAMP Sepharose, the cGMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from tissues of the prawn Palaemon adspersus was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The degree of enzyme purification was 11 200, recovery--6.5%; the isoelectric point for the enzyme lies at 5.5. Data from gel filtration and centrifugation in sucrose density gradient suggest that the dimer of cGMP-dependent protein kinase has a molecular weight of 157 000, sedimentation coefficient of 7.2S and a Stokes' radius of 50 A. An active form of the enzyme with Mr = 76 500 (4.5S, 39 A) which apparently represents a subunit of the cGMP-dependent protein kinase was discovered. The activity of the both enzyme forms are stimulated by low concentrations of cGMP (Ka = 1.10(-7) M). The monomer and dimer molecules appear as prolate ellipsoids with axial ratios close to 7. The native cGMP-dependent protein kinase is probably made up of two subunits each of which contains a regulatory and a catalytic sites.     

The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study.

The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.     

Hydrodynamic properties of macromolecular complexes. I. Translation

We have developed an improved theory for calculating the translational frictional coefficients of rigid macromolecular complexes composed of unequal spherical subunits. The Yamakawa hydrodynamic interaction tensor, which improves on the Oseen tensor by taking account of the finite sizes of the frictional subunits, has been generalized to accomodate nonidentical subunits. Iterative numerical methods are described for solving the set of simultaneous hydrodynamic interaction equations, thus avoiding preaveraging. The theory is applied to prolate ellipsoids of revolution, to lollipops, and to dumbbells, and comparison is made with earlier, more approximate theories.     

Shape of Ocr, the gene 0.3 protein of bacteriophage T7: modeling based on light scattering experiments

Ocr, the first protein expressed by bacteriophage T7, inhibits type Iota DNA restriction enzymes by preventing them from binding to DNA. This inhibition allows the phage to successfully infect the host. The shape of ocr is modeled on the basis of static and dynamic light scattering measurements. The static light scattering data confirm previous observations that ocr exists in solution as a dimer. The diffusion constant determined by dynamic light scattering indicates a nonspherical shape of the ocr dimer. Hydrodynamic models of ellipsoids are presented, and it is argued that ocr is best described by a prolate ellipsoid with dimensions of 10.4 nm by 2.6 nm. The size and shape predicted by this model are consistent with ocr acting as a mimic of the DNA structure bound by type Iota restriction enzymes.