Computation of the sedimentation coefficient,s20,w0, from the analytical ultracentrifuge run by an interactive program on the Apple II microcomputer

We have developed a computer program for the calculation of the sedimentation coefficient of a macromolecule from the raw data collected from an analytical ultracentrifuge run. The program is written for the Apple II microcomputer and is capable of calculating the sedimentation coefficient at an infinite dilution from the data collected at varying concentrations.     

FUGE: an analytical ultracentrifuge simulation for teaching purposes

An IBM-compatible microcomputer program for teaching purposesis described which simulates the operation of a sedimentationvelocity determination of a protein in an analytical ultracentrifugeusing schlieren optics. The program operates in speeded-up timeand simulates the major procedures which would need to be carriedout to operate such an instrument. The position of the sedimentingboundary can be observed at any time during the run, and upto six ‘photographs’ can be recorded for subsequentanalysis. Calculation of sedimentation coefficient, diffusioncoefficient and mol. wt can be made from a dot-matrix printout.Ten representative proteins are stored within the program, butprovision exists for user-supplied data. Received on June 25, 1987; accepted on September 9, 1987     

Determination of the sedimentation coefficient distribution by least-squares boundary modeling

A new method is presented for the calculation of apparent sedimentation coefficient distributions g*(s) for the size-distribution analysis of polymers in sedimentation velocity experiments. Direct linear least-squares boundary modeling by a superposition of sedimentation profiles of ideal nondiffusing particles is employed. It can be combined with algebraic noise decomposition techniques for the application to interference optical ultracentrifuge data at low loading concentrations with significant systematic noise components. Because of the use of direct boundary modeling, residuals are available for assessment of the quality of the fits and the consistency of the g*(s) distribution with the experimental data. The method can be combined with regularization techniques based on F statistics, such as used in the program CONTIN, or alternatively, the increment of s values can be adjusted empirically. The method is simple, has advantageous statistical properties, and reveals precise sedimentation coefficients. The new least-squares ls-g*(s) exhibits a very high robustness and resolution if data acquired over a large time interval are analyzed. This can result in a high resolution for large particles, and for samples with a high degree of heterogeneity. Because the method does not require a high frequency of scans, it can also be easily used in experiments with the absorbance optical scanning system. Published 2000 John Wiley & Sons, Inc.     

Sedimentation coefficient of polyoma virus DNA

The sedimentation coefficient of the twisted circular form of polyoma virus DNA is calculated from the Kirkwood sedimentation–diffusion equation, the structure being assumed to be a rigid double superhelix. Agreement with the experimental sedimentation coefficient can be obtained, with the use of an experimental value for the number of superhelical turns, when the pitch of the superhelix is intermediate between its minimal and maximal possible values. Another model, which has been proposed for polyoma DNA at low ionic strengths, may be visualized as a superhelical structure wound about a torus. Calculations of sedimentation coefficients for this model agree qualitatively with experimental data at ionic strengths Below 10^?2M.     

Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation

Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The sedimentation coefficient distribution c(s) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the sedimentation coefficient consistent with the traditional s approximately M(2/3) power law. It provides a high hydrodynamic resolution, where diffusional broadening of the sedimentation boundaries is deconvoluted from the sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of sedimentation coefficient and frictional ratio, c(s,f(r)), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c(M,R(S)), and sedimentation coefficient-molar mass distributions c(s,M). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.     

Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery

Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 +/- 0.35 X 10(-7) cm2/s, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio less than or equal to 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was much greater than 10 micron in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 microM MgCl2 at 4 degrees C. On the basis of the diffusion coefficient, we conclude that monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.     

Calculation of s20,w values using ultracentrifuge sedimentation data from linear sucrose gradients, an improved, simplified method.

A mathematical method is described for calculating the sedimentation coefficient (S20,W) with ultracentrifuge data from linear sucrose gradients. Gradient density and viscosity functions are precisely described by regression equations, which permit continuous evaluation (by integration) of the effects of gradient geometry on particle sedimentation. The results agree with previously used and more complex methods.     

The Sedimentation Pattern of D-Glucose-6-Phosphate Dehydrogenase from Bovine Fetal and Adult Erythrocytes

Erythrocytes from bovine fetuses contain about 2.4 times higher D-glucose-6-phosphate dehydrogenase activities than erythrocytes from adult cows and bulls. Studying whether this is due to the existence of a special fetal type of enzyme or an increased amount of enzyme in fetal erythrocytes, the sedimentation coefficients of the enzymes have been estimated by s-zonal ultracentrifugation, and compared to normal and deficient human erythrocyte D-glucose-6-phosphate dehydrogenase, s-zonal ultracentrifugations have been performed with a computer optimized isokinetic sucrose gradient. The mainlines in the program used for calculation of sedimentation coefficients are described. Bovine fetal and adult erythrocyte D-glucose-6-phosphate dehydrogenase was found to have the same sedimentation coefficient of 7.4 S which is different from the sedimentation coefficient of 6.4 S of both human types of the enzyme. The sedimentation coefficients of 6-phospho-D-gluconate dehydrogenase from bovine fetal, bovine adult and human erythrocytes were 6 S for all three types of this enzyme. By cellulose acetate electrophoresis bovine fetal and adult D-glucose-6-phosphate dehydrogenase show the same mobility, again differing from the normal and deficient human type. The results of these experiments show that bovine fetal and adult erythrocytic D-glucose-6-phosphate dehydrogenase with respects to molecular parameters are closely related and perhaps identical enzymes.     

Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.     

A comparison of weight average and direct boundary fitting of sedimentation velocity data for indefinite polymerizing systems

Analysis of sedimentation velocity data for indefinite self-associating systems is often achieved by fitting of weight average sedimentation coefficients (s(20,w)) However, this method discriminates poorly between alternative models of association and is biased by the presence of inactive monomers and irreversible aggregates. Therefore, a more robust method for extracting the binding constants for indefinite self-associating systems has been developed. This approach utilizes a set of fitting routines (SedAnal) that perform global non-linear least squares fits of up to 10 sedimentation velocity experiments, corresponding to different loading concentrations, by a combination of finite element simulations and a fitting algorithm that uses a simplex convergence routine to search parameter space. Indefinite self-association is analyzed with the software program isodesfitter, which incorporates user provided functions for sedimentation coefficients as a function of the degree of polymerization for spherical, linear and helical polymer models. The computer program hydro was used to generate the sedimentation coefficient values for the linear and helical polymer assembly mechanisms. Since this curve fitting method directly fits the shape of the sedimenting boundary, it is in principle very sensitive to alternative models and the presence of species not participating in the reaction. This approach is compared with traditional fitting of weight average data and applied to the initial stages of Mg(2+)-induced tubulin self-associating into small curved polymers, and vinblastine-induced tubulin spiral formation. The appropriate use and limitations of the methods are discussed.     

Crystallohydrodynamics of protein assemblies: Combining sedimentation, viscometry, and x-ray scattering

Crystallohydrodynamics describes the domain orientation in solution of antibodies and other multidomain protein assemblies where the crystal structures may be known for the domains but not the intact structure. The approach removes the necessity for an ad hoc assumed value for protein hydration. Previous studies have involved only the sedimentation coefficient leading to considerable degeneracy or multiplicity of possible models for the conformation of a given protein assembly, all agreeing with the experimental data. This degeneracy can be considerably reduced by using additional solution parameters. Conformation charts are generated for the three universal (i.e., size-independent) shape parameters P (obtained from the sedimentation coefficient or translational diffusion coefficient), nu (from the intrinsic viscosity), and G (from the radius of gyration), and calculated for a wide range of plausible orientations of the domains (represented as bead-shell ellipsoidal models derived from their crystal structures) and after allowance for any linker or hinge regions. Matches are then sought with the set of functions P, nu, and G calculated from experimental data (allowing for experimental error). The number of solutions can be further reduced by the employment of the D max parameter (maximum particle dimension) from x-ray scattering data. Using this approach we are able to reduce the degeneracy of possible solution models for IgG3 to a possible representative structure in which the Fab domains are directed away from the plane of the Fc domain, a structure in accord with the recognition that IgG3 is the most efficient complement activator among human IgG subclasses.     

Calculation of s20,w values using ultracentrifuge sedimentation data from linear sucrose gradients,an improved,simplified method

A mathematical method is described for calculating the sedimentation coefficient (s_{20, w}) with ultracentrifuge data from linear sucrose gradients. Gradient density and viscosity functions are precisely described by regression equations, which permit continuous evalution (by integration) of the effects of gradient geometry on particle sedimentation. The results agree with previously used and more complex methods.     

Isolation and some properties of components of nuclear polyhedra from the cabbage looper, Trichoplusia ni

Nuclear polyhedra obtained from diseased cabbage looper, Trichoplusia ni, were digested with sodium carbonate-saline buffer, pH 11.0. The dissolved polyhedra formed 3 general zones when subjected to density gradient centrifugation. The slowest sedimenting component (Zone 1) had an ultraviolet absorption curve typical of protein and a sedimentation coefficient of 11 S. Capsids, 310 × 40 nm, were located in Zone 2. Virus particles were found in 1–3 bands (Zone 3); those with envelopes measured 300 × 72 nm, and those without envelopes measured 300 × 33 nm. Virus preparations stained with phosphotungstic acid at pH 7.0 exhibited extensive disruption whereas preparations stained at pH 3.0 did not. Virus particles in the sodium carbonate-saline-digested polyhedra had a sedimentation coefficient of 1228 S. Virus particles isolated by high speed centrifugation had a sedimentation coefficient of 1530 S.     

A convenient program for the rapid calculation of sedimentation coefficients in linear salt or sucrose gradients

A program is presented for calculating s_20,w values from data obtained by zone sedimentation in linear sucrose or salt gradients in a variety of rotors at temperatures ranging from 0° to 20°C. The program can be either run with the use of high-speed computers or performed with the aid of a small calculator in a reasonably short time.     

Enhanced resolution of sedimentation coefficient distribution profiles by extrapolation to infinite time

Sedimentation velocity experiments can be used to identify two or more independent non-interacting macromolecules, which differ in their size by only a few percent. The procedure requires the extrapolation of differential apparent sedimentation coefficient distributions obtained at different running time to t → ∞ and works because it eliminates or greatly reduces diffusion effects. Here, we present an improved time extrapolation function of sedimentation distribution profiles originally presented by Stafford (In: Harding, Rowe, Horton (eds.) Analytical ultracentrifugation in biochemistry and polymer science, 1992). We describe a computing procedure with the program lamm to analyze concentration profiles obtained by absorbance or interference optics that utilizes suitable smoothing methods for noisy data sets and present examples which include time invariant noises.     

Chromatographic Separation, and Characteristics of Nucleic Acids from HeLa Cells

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s₂₀^o,w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained.     

Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation

This study explores the capability of modern analytical ultracentrifugation (AUC) to characterize the homogeneity, under product formulation conditions, of preparations of adenovirus vectors used in gene therapy and to assess the lot-to-lot consistency of this unique drug product. We demonstrate that a single sedimentation velocity run on an adenovirus sample can detect and accurately quantify a number of different forms of virus particles and subvirus particles. These forms include (a) intact virus monomer particles, (b) virus aggregates, (c) empty capsids (ECs), and (d) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly (or as a result of damage to the intact adenovirus or EC material during all phases of virus production). This information, which is collected on adenovirus samples under the exact formulation conditions that exist in the adenovirus vial, is obtained by direct boundary modeling of the AUC data generated from refractometric and/or UV detection systems using the computer program SEDFIT developed by Peter Schuck. Although both detectors are useful, refractometric detection using the Rayleigh interferometer offers a key advantage for providing accurate concentration information due to the similar response factors for both protein and DNA and its insensitivity to light scattering effects. Additional AUC data obtained from analytical band sedimentation velocity and density gradient sedimentation equilibrium experiments in CsCl with UV detection were also generated. These results further support conclusions concerning the solution properties of adenovirus, the identity of the different virus species, and the overall capability of boundary sedimentation velocity analysis.     

19 A solution structure of the filarial nematode immunomodulatory protein,ES-62

ES-62, a protein secreted by filarial nematodes, parasites of vertebrates including humans, has an unusual posttranslational covalent addition of phosphorylcholine to an N-type glycan. Studies on ES-62 from the rodent parasite Acanthocheilonema viteae ascribe it a dominant role in ensuring parasite survival by modulating the host immune system. Understanding this immunomodulation at the molecular level awaits full elucidation but distinct components of ES-62 may participate: the protein contributes aminopeptidase-like activity whereas the phosphorylcholine is thought to act as a signal transducer. We have used biophysical and bioinformatics-based structure prediction methods to define a low-resolution model of ES-62. Sedimentation equilibrium showed that ES-62 is a tightly bound tetramer. The sedimentation coefficient is consistent with this oligomer and the overall molecular shape revealed by small angle x-ray scattering. A 19 A model for ES-62 was restored from the small-angle x-ray scattering data using the program DAMMIN which uses simulated annealing to find a configuration of densely packed scattering elements consistent with the experimental scattering curve. Analysis of the primary sequence with the position-specific iterated basic local alignment search tool, PSI-BLAST, identified six closely homologous proteins, five of which are peptidases, consistent with observed aminopeptidase activity in ES-62. Differences between the secondary structure content of ES-62 predicted using the consensus output from the secondary structure prediction server JPRED and measured using circular dichroism are discussed in relation to multimeric glycosylated proteins. This study represents the first attempt to understand the multifunctional properties of this important parasite-derived molecule by studying its structure.     

Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile.

A procedure is described for computing sedimentation coefficient distributions from the time derivative of the sedimentation velocity concentration profile. Use of the time derivative, (delta c/delta t)r, instead of the radial derivative, (delta c/delta r)t, is desirable because it is independent of time-invariant contributions to the optical baseline. Slowly varying baseline changes also are significantly reduced. An apparent sedimentation coefficient distribution (i.e., uncorrected for the effects of diffusion), g*(s), can be calculated from (delta c/delta t)r as [formula: see text] where s is the sedimentation coefficient, omega is the angular velocity of the rotor, c0 is the initial concentration, r is the radius, rm is the radius of the meniscus, and t is time. An iterative procedure is presented for computing g*(s)t by taking into account the contribution to (delta c/delta t)r from the plateau region to give (delta c/delta t)corr. Values of g*(s)t obtained this way are identical to those of g*(s) calculated from the radial derivative to within the roundoff error of the computations. Use of (delta c/delta t)r, instead of (delta c/delta r)t, results in a significant increase (greater than 10-fold) in the signal-to-noise ratio of data obtained from both the uv photoelectric scanner and Rayleigh optical systems of the analytical ultracentrifuge. The use of (delta c/delta t)r to compute apparent sedimentation coefficient distributions for purposes of boundary analysis is exemplified with an antigen-antibody system.     

Physical properties, limited proteolysis, and acetylation of tyrosine aminotransferase from rat liver

The native and one of the modified forms of tyrosine aminotransferase were purified from rat liver and characterized. Several hydrodynamic properties of the native enzyme are: Stokes radius, 46 A; subunit isoelectric point, 5.6; sedimentation coefficient, 5.6 S, frictional ratio, 1.44; diffusion coefficient, 4.65 X 10(-7) cm2 s-1; extinction coefficient of a 1% solution (w:v) at 280 nm, 10.5 cm-1. The molecular weight of the dimeric protein is 110,500 as calculated from the Stokes radius and sedimentation coefficient. The subunit of the modified form is of lower molecular weight than the subunit of the native enzyme and has a pI of about 5.9. During isoelectric focusing, both forms of the enzyme separate into two components. The more acidic component that is resolved from the native enzyme is phosphorylated, but the other component is not. The amino acid composition of native tyrosine aminotransferase differs from values reported for mixtures of the three forms of this enzyme. Neither the native nor the modified forms of the enzyme possess a free alpha-amino group as judged by dansylation, nor can they be digested with leucine aminopeptidase, implying that the NH2-terminus is blocked. The possibility that tyrosine aminotransferase is acetylated was examined by translating poly(A)+RNA from hepatoma cells in a cell-free translational system in the presence and absence of inhibitors of protein acetylation. [35S]Tyrosine aminotransferase synthesized in the presence of the inhibitors has a more basic isoelectric point than the native enzyme as determined by isoelectric focusing, suggesting that the enzyme is acetylated either at the NH2-terminal or the epsilon-amino group of an internal lysine. When digested by either of two lysosomal proteases, tyrosine aminotransferase is cleaved to a smaller size. These data show that tyrosine aminotransferase is susceptible to several post-translational modifications.