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PubMed1980

Turbidity measurements in an analytical ultracentrifuge. Determinations of mass per length for filamentous viruses fd, Xf, and Pf3.

An analytical ultracentrifuge has been used to measure light-scattering intensities by the transmittance method. The technique, which is applicable to particles of many sizes and shapes, has the principal advantage that samples can be kept free of dust during the measurements. Also, sample volumes are small, and the scanner and interference optics can be used simultaneously to obtain, for a given sedimenting boundary, turbidity steps at different wavelengths and the concentration step. In the present application the data yield mass per length estimates for three filamentous viruses, 19 100 daltons/nm for fd, 19 600 daltons/nm for Pf3, and 19 100 daltons/nm for Xf.

Coliphages↗
PubMed1976

Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Active enzyme ultracentrifugation studies.

Active enzyme ultracentrifugation studies of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1.) show that the enzymatic reaction is catalyzed by a molecular species characterized by an S20,W value of about 34 S, whatever the reduced substrate may be (tri- or diphosphopyridine nucleotide). The filamentous aggregated form of the enzyme (S20,W = 121 S and higher), identified by previous investigations (Cohen, P. T., and Kaplan, N. O. (1970), J. Biol. Chem. 245, 2825-2836; Louie, D. D., Kaplan, N. O., and Mc Lean, J. D. (1972), J. Mol. Biol. 70, 651-664), appears, therefore, to be an inactive species. The physiological implications of the enzyme are discussed. Several lines of evidence lead to the conclusion that the transhydrogenase might act as an essential link between carbohydrate catabolism and the respiratory chain.

Macromolecular Substances↗
PubMed2001

Analytical ultracentrifugation studies of translin: analysis of protein-DNA interactions using a single-stranded fluorogenic oligonucleotide.

Translin is a recently identified nucleic acid binding protein that appears to be involved in the recognition of conserved sequences found at many chromosomal breakpoints. Previous reports indicate that, based on gel filtration analysis and electron microscopy of protein-DNA complexes, translin forms an octameric structure that binds the DNA. In this study, we further examine the possibility of self-association of translin and its interactions with DNA by analytical ultracentrifugation. Sedimentation velocity analysis of translin indicates that the predominant species sediments with a sedimentation coefficient of 8.5 S and has a frictional ratio, f/f(omicron), of 1.35; these data are consistent with the presence of an octamer with an ellipsoidal configuration; a small amount of a component with significantly higher mass is also present. Equilibrium sedimentation studies of translin at three different protein concentrations also indicate that the predominant species present is an octamer with a minor fraction of aggregated species. Neither monomer nor dimer was detected. Sedimentation equilibrium studies of translin with an FITC-labeled single-stranded oligonucleotide were performed to examine the interaction. A novel analysis method has been developed to analyze protein-nucleic acid interactions based on global fitting of scans of 280 and 490 nm to appropriate mathematical models. Utilizing this method, it was determined that the DNA binding species of translin is an octamer binding a single-stranded oligonucleotide with a DeltaG degrees value of -9.49 +/- 0.12 kcal/mol, corresponding to a dissociation constant, K(d), of 84 +/- 17 nM. On the basis of this evidence and electron microscopy, it is envisioned that translin forms an annular structure of eight subunits, hydrodynamically an oblate ellipsoid, which binds DNA at chromosomal breakpoints.

DNA, Single-Stranded↗
PubMed2003

Dimeric structure of the six-domain VibF subunit of vibriobactin synthetase: mutant domain activity regain and ultracentrifugation studies.

Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide sythases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. Previous data on FAS and PKS subunits have indicated that they are homodimers and that some of their catalytic functions can work in trans. When NRPS assembly lines have been probed for comparable formation of stable oligomers, no evidence had been forthcoming that species other than monomer forms were active. In this work we focus on the six-domain (Cy1-Cy2-A-C1-PCP-C2) enzyme VibF from the vibriobactin synthetase assembly line, which contains three other proteins, VibB, VibE, and VibH, that--when purified and mixed with VibF and the substrates ATP, threonine, 2,3-dihydroxybenzoate (DHB), and norspermidine--produce the iron chelator vibriobactin. Using a deletion of the Cy1 domain and separate inactivating mutations in the Cy2, A, PCP, and C2 domains of VibF, we report regain of catalytic activity upon mutant protein mixing that argues for heterodimer formation, stable for hundreds to thousands of catalytic cycles, with acyl chain processing and transfer around blocked domains. Ultracentrifugation data likewise confirm a dimeric structure for VibF and establish that domains within NRPS dimeric modules can act on acyl chains in trans. The results described here are the first indication for an NRPS subunit that homodimerization can occur and that there is a continuum of functional oligomerization states between monomers and dimers in nonribosomal peptide synthetases.

Acylation↗
PubMed2004

pKa and aggregation of bilirubin: titrimetric and ultracentrifugation studies on water-soluble pegylated conjugates of bilirubin and fatty acids.

A water-soluble conjugate (1) with intact carboxyl groups was prepared by addition of poly(ethylene glycol) thiol (MPEG-SH) regiospecifically to the exo vinyl group of bilirubin. (1)H and (13)C NMR and absorbance spectroscopy in CDCl(3) and DMSO-d(6) confirmed the assigned structure and showed that pegylation did not disrupt the hydrogen-bonded ridge-tile conformation of the pigment moiety. Aqueous solutions of 1 were optically clear, but NMR signals were seen only from the MPEG portion and none from the tetrapyrrole, consistent with dissolved assemblies containing aggregated bilirubin cores within mobile polyether chains. On alkalinization (pH >12), signals from the pigment moiety reappeared. Titrimetric measurements on 1 in water showed the pK(a)'s of the two carboxyl groups to be similar (average 6.42). Control studies with pegylated half-esters of succinic, suberic, brassylic, thapsic, and 1,20-eicosanedioic acid showed that pegylation per se has little, if any, effect on carboxyl ionization. However, aggregation increases the apparent pK(a) by approximately 1-2 units. The molecularity of bilirubin in solution was further characterized by ultracentrifugation. Over the pH range 8.5-10 in buffer, bilirubin formed multimers with aggregation numbers ranging from approximately 2-7. Bilirubin is monomeric in DMSO or CHCl(3) at approximately 2 x 10(-)(5) M, but aggregation occurred when the CHCl(3) was contaminated with trace adventitious (perhaps lipoidal) impurities. These observations show that aggregation increases the pK(a)'s of aliphatic carboxylic acids relative to their monomer values in water. They are consistent with earlier (13)C NMR-based estimates of approximately 4.2 and approximately 4.9 for the aqueous pK(a)'s of bilirubin and similar studies of bilirubin in micellar bile-salt solutions. Together with earlier work, they confirm that the pK(a)'s of bilirubin are about normal for aliphatic carboxyls and suggest that the high (>7.5) values occasionally reported, including those based on CHCl(3) partitioning, are artifacts of aggregation or technique.

Bilirubin↗
PubMed2005

Influence of temperature on the conformation of canine plasminogen: an analytical ultracentrifugation and dynamic light scattering study.

Plasminogen is known to undergo an extremely large conformational change when it binds ligands; the two well-established conformations are either closed (absence of external ligand) or open (presence of external ligand). We show here that plasminogen is more complicated than can be accommodated by a two-state, closed/open, model. Temperature changes induce large structural changes which can be detected with either dynamic light scattering or analytical ultracentrifugation. The temperature-induced changes are not related to the classical closed/open conformational change since both closed and open forms of the protein are similarly influenced. It appears as though the packing density of the protein increases as the temperature is raised. Over the range 4-20 degrees C, the Stokes' radius of the classical closed plasminogen goes from 4.7 to 4.2 nm, and that of the classical open form goes from 5.55 to 5.0 nm. These changes in packing can be rationalized if temperature change induces a large conformational change and if this is accompanied by a large change in hydration, by a change in solute binding, or by a change in the total void volume of the protein.

Animals↗
PubMed1998

Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation.

Analytical ultracentrifugation methods were utilized to further characterize the acid denaturation pathways of wild-type, V30M, and L55P transthyretin (TTR) that generate intermediates leading to amyloid fibril formation and possibly the diseases senile systemic amyloidosis and familial amyloid polyneuropathy. Equilibrium and velocity methods were employed herein to characterize the TTR quaternary structural requirements for amyloid fibril formation. From neutral to slightly acidic conditions (pH 7.5-5.1), wild-type transthyretin (0.2-0.3 mg/mL, 100 mM KCl, 37 degrees C) exists as a tetramer and is incapable of fibril formation. Under more acidic conditions (pH 5 to 3.9), tetrameric wild-type TTR slowly dissociates to a monomer having an alternatively folded tertiary structure(s) that self-assembles at physiological concentration (0.2 mg/mL) into a ladder of quaternary structural intermediates of increasing molecular weight. These intermediates appear to be on the pathway of amyloid fibril formation, since they ultimately disappear when amyloid fibrils are observed. The V30M and L55P TTR variants exhibit similar acid denaturation pathways, with the exception that dissociation of the tetramer to the monomeric amyloidogenic intermediate occurs at a higher pH and to a much greater extent, allowing the quaternary structural intermediates to be readily observed by velocity methods. Partial denaturation and assembly of the monomeric amyloidogenic intermediate(s) occur at pH 5.4 for V30M and L55P TTR over a 72 h period, during which wild-type TTR maintains its normal tetrameric three-dimensional structure. Interestingly, the L55P and V30M familial amyloid polyneuropathy (FAP) associated variants form amyloid protofilaments at pH 7.5 (37 degrees C) after several weeks of incubation, suggesting that the activation barriers for TTR tetramer dissociation to the monomeric amyloidogenic intermediate are much lower for the FAP variants relative to wild-type TTR, which does not form amyloid or amyloid protofilaments under these conditions. This study establishes the key role of the monomeric amyloidogenic intermediate and its self-assembly into a ladder of quaternary structural intermediates for the formation of wild-type, V30M, and L55P transthyretin amyloid fibrils.

Amyloid↗
PubMed2000

X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery.

Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 A crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated.

Computer Simulation↗
PubMed2000

Further observations on the size, shape, and hydration of casein micelles from novel analytical ultracentrifuge and capillary viscometry approaches.

The size, shape, and hydration of casein micelles were estimated using a combination of sedimentation velocity (time-derivative analysis) in the analytical ultracentrifuge and capillary viscometry applied to skimmed milk. On the basis of sedimentation time-derivative and Wales-van Holde analyses the casein micelles appear as large spherical molecules of s0T,b = 845S, Mw approximately 2.8 x 10(8), hydrodynamic radius approximately 77.8 nm, and ks/[eta] = 1.6. The molecular hydration (i.e., the extent of chemically bound and physically entrained solvent) was calculated to be 3.4 g/g. These results appear to be in good agreement with comparable results from electron microscopy and dynamic light scattering.

Algorithms↗
PubMed2006

Pressure cell assisted solution characterization of galactomannans. 3. Application of analytical ultracentrifugation techniques.

The pressure heating cell approach previously applied to galactomannans in two earlier studies is now used to prepare samples for characterization using the analytical ultracentrifuge. Sedimentation velocity data were obtained for both guar gum and locust bean gum samples. These were compared to our earlier light scattering and intrinsic viscosity measurements on samples prepared using identical temperature and pressure profiles. A number of methods were then employed to obtain chain persistence lengths, including the Hearst-Stockmayer and Bohdanecky wormlike chain approaches. These results were compared to earlier results obtained using methods appropriate for excluded volume coil and rodlike chains, respectively.

Carbohydrate Sequence↗
PubMed2005

Analytical ultracentrifugation for characterizing nanocrystals and their bioconjugates.

Analytical ultracentrifugation (AU) provides a general way to probe the polydispersity of nanoparticles and the formation of bioconjugates in solution. Unconjugated gold nanocrystals show sedimentation coefficient distributions that are in agreement with size distributions as measured by TEM. AU is sensitive to the size/shape changes elicited by conjugation, in this case to lactose repressor (LacI). AU data reveal saturating protein concentrations for conjugates that correspond to the measured stoichiometry of the complex under these conditions.

Algorithms↗
PubMed1999

Investigation of protein-surfactant interactions by analytical ultracentrifugation and electron paramagnetic resonance: the use of recombinant human tissue factor as an example.

PURPOSE: The purpose of this work is to utilize electron paramagnetic resonance (EPR) spectroscopy in conjunction with analytical ultracentrifugation (AUC) to investigate the binding of surfactants to proteins with a transmembrance domain. As an example these methods have been used to study the interaction of a nonionic surfactant, C12E8, to recombinant human tissue factor (rhTF) in liquid formulations. The complementary nature of the two techniques aids in data interpretation when there is ambiguity using a single technique. In addition to binding stoichiometries, the possibility of identifying the interacting domains by using two forms of rhTF is explored. METHODS: Two recombinant, truncated forms of human tissue factor were formulated in the absence of phospholipids. Neither of the recombinant proteins, produced in E. coli, contains the cytoplasmic domain. Recombinant human tissue factor 243 (rhTF 243) consists of 243 amino acids and includes the transmembrane sequences. Recombinant human tissue factor 220 (rhTF 220), however, contains only the first 221 amino acids of the human tissue factor, lacking those of the transmembrane region. EPR and AUC were used to investigate the interactions between these two forms of rhTF and polyoxyethylene 8 lauryl ether, C12E8. RESULTS: Binding of C12E8 to rhTF 243 is detected by both EPR spectroscopy and AUC. Although a unique binding stoichiometry was not determined, EPR spectroscopy greatly narrowed the range of possible solutions suggested by the AUC data. Neither technique revealed an interaction between rhTF 220 and C12E8. CONCLUSIONS: The complementary nature of EPR spectroscopy and AUC make the combination of the two techniques useful in data interpretation when studying the interactions between rhTF and C12E8. By utilizing these techniques in this study, the binding stoichiometry of rhTF 243 to C12E8 ranges from 1.2:1 to 1.3:0.6 based on an aggregation number of 120. This binding is consistent with previously reported activity data that showed an increase in clotting rate when rhTF 243 is in the presence of C12E8 micelles. From the rhTF 220 data, it can further be concluded that the transmembrane domain of rhTF is necessary for interactions with C12E8.

Electron Spin Resonance Spectroscopy↗
PubMed1967

The separation of vanillate O-demethylase from protocatechuate 3,4-oxygenase by ultracentrifugation.

1. Protocatechuate 3,4-oxygenase in the soluble part of a cell-free extract of Pseudomonas fluorescens (strain T) sedimented more rapidly than vanillate O-demethylase under specified conditions in a preparative ultracentrifuge. 2. The supernatant from this process contained vanillate O-demethylase and formaldehyde dehydrogenase, and when supplemented with NADH oxidized vanillate with an uptake of 1 mole of oxygen/mole of substrate and accumulation of protocatechuate. 3. This uptake was decreased to 0.5mole/mole of substrate in the presence of semicarbazide as trapping agent for formaldehyde. 4. Reasons are presented for the process of methyl group removal from vanillate being oxidative demethylation.

Cell-Free System↗
PubMed1971

Protein aggregation in C-phycocyanin. Studies at very low concentrations with the photoelectric scanner of the ultracentrifuge.

Solutions of C-phycocyanin of very low concentrations were examined by sedimentation-velocity studies in the Spinco model E ultracentrifuge equipped with a photoelectric scanning system and a monochromator. At sufficiently low concentrations complete disaggregation from the hexamer to the monomer was observed. The equilibrium constant of monomer to hexamer was estimated to be approx. 10(30). For studies of aggregation over the complete range of concentration, C-phycocyanins from Phormidium luridum and Lyngbya sp. were used. Sedimentation-velocity studies at high concentration with schlieren optics are reported for C-phycocyanins from Anabaena variabilis and Lyngbya sp. The pH-dependence of aggregation and the temperature-dependence of trimer-hexamer equilibrium for phycocyanins from these algae were found to be similar to those of other C-phycocyanins. The principal feature of the pH-dependence is the dominance of hexamers at the isoelectric point. Increasing temperature increased the amount of hexamer and decreased the amount of trimer.

Centrifugation, Density Gradient↗
PubMed1972

The statistical estimation of molecular weights from normal and difference ultracentrifuge boundaries.

1. To determine molecular weights from boundary data taken from a sedimentation velocity experiment in an ultracentrifuge, the parameter s/D must be estimated. This can be obtained by using non-linear statistical methods to fit a mathematical model [the Fujita & MacCosham (1959) equation] to the results. 2. The statistical method chosen was the simplex method of Nelder & Mead (1965), which was found to be ideal for this problem. Internal errors were calculated at the end of the search for the minimum in the residuals, but in general these errors were found to not represent the overall true error of the experiment. 3. Calculations of molecular weights of myoglobin showed that instabilities at low concentrations of protein (less than 0.8mg/ml) disturbed the calculation of s/D. If 1% (w/v) sucrose was included in the solvent, these instabilities were decreased, and extrapolating to infinite time the linear function of s versus 1/(time) gave an acceptable value for s with an error of +/-4.8%. The estimates of the molecular weights were less well-defined and the mean value was low by 8%, with an estimated error of the mean of +/-3%. The conclusion was that vibration was responsible for the instabilities without sucrose. 4. The Fujita-MacCosham equation can be extended to make it possible to estimate ratios of sedimentation and molecular weights for difference boundaries. Tests using two solutions of orosomucoid in which a 2% decrease in velocity of one boundary was achieved by adding a calculated quantity of sucrose showed that the analysis gave realistic values for the two ratios, and the error for the ratio of sedimentation coefficients was +/-10%. The error was larger for the estimated ratio of the molecular weights, but the analysis gave the expected value for the ratio.

Glycoproteins↗
PubMed1977

A comparison of the association of yeast phosphoglycerate mutase (EC2.7.5.3) with that of haemoglobin. An ultracentrifuge study.

1. Previous work showed that yeast phosphoglycerate mutase (EC 2.7.5.3) has a mol.wt. of between 107000 and 110000. Preliminary examination showed that at dilutions less than 0.1 g/1 the enzyme dissociated into its subunits. 2. This dissociation was quantitatively examined by both equilibrium and velocity centrifugation. 3. The mathematical analysis of the equilibrium records was tested against oxyhaemoglobin in a variety of ionic strengths and at two temperatures. 4. The estimated L2,4 (interaction coefficient) for oxyhaemoglobin generally agreed with published values except at 6 degrees C in 0.9 M-NaCl, when it was 2.5 times larger than the published value. 5. Statistical analysis of ultracentrifugal-equilibrium experiments showed that the predominant reaction for phosphoglycerate mutase was monomer in equilibrium tetramer, to give an L1,4 of 40.3+/-23.4 (S.D.)1(3)-g(-3) at 20 degrees C. Decreasing the temperature decreased the association to given an enthalpy of between 40 and 60kJ/mol. 6. Analysis of velocity experiments carried out with concentrations varying from 0.3 to 17 g/1 gave an L1,4 of 3111(3)-g(-3). Incorporating errors from estimating S20,w into the analysis showed that this estimate could range from 893 to 1421(3)-g(-3). 7. The concentration-dependence of S20,w was 0.95 litre-g-1 and s020,w for the tetramer was 66.9ps. 8. These results are discussed in relation to the activity of the enzyme.

Hemoglobins↗
PubMed1989

Subunit interactions in Propionibacterium shermanii methylmalonyl-CoA mutase studied by analytical ultracentrifugation.

The effect of increasing ionic strength on adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii was studied by using analytical ultracentrifugation. Both sedimentation-velocity and low-speed sedimentation-equilibration measurements show that the enzyme dissociates progressively into its two dissimilar subunits with increasing ionic strength. Equilibrium between the alpha beta-dimer and the separated subunits is rapidly established under these conditions. Dissociation is accompanied by loss of enzymic activity, but the position of the equilibrium is unaffected by the presence of either substrate or adenosylcobalamin cofactor.

Acyl Coenzyme A↗
PubMed1993

Methylmalonyl-CoA mutase from Propionibacterium shermanii: characterization of the cobalamin-inhibited form and subunit-cofactor interactions studied by analytical ultracentrifugation.

A large proportion of adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermannii is isolated in an inactive form which contains a tightly bound cobalamin. Even when the enzyme was denatured in 5.0 M guanidine hydrochloride the cobalamin remained associated with the protein. However, when dithiothreitol was added to the denatured protein, the pink inhibitor was rapidly converted into a yellow-brown compound which could be removed by dialysis. Enzyme activity could be recovered after removal of the denaturant, although surprisingly this did not depend on prior treatment with dithiothreitol. The interaction between the protein and inhibitor was investigated by using analytical ultracentrifugation under denaturing conditions. The sedimentation coefficient s20,w was measured in various concentrations of guanidine hydrochloride. A complicated picture emerged in which at low denaturant concentrations subunit dissociation, partial unfolding and aggregation occur, whereas at high concentration the protein behaves as a monodisperse species. No major differences in sedimentation were observed between the enzyme-cobalamin complex and the cobalamin-free enzyme, suggesting that the inhibitor does not significantly stabilize higher-order structure within the protein.

Dithiothreitol↗