Salt-dependent interconversion of inner histone oligomers.

The inner histone complex, extracted from chicken erythrocyte chromatin in 2 M NaCL AT pH 7.4, has been characterized by sedimentation equilibrium and sedimentation velocity. High speed sedimentation equilibrium studies indicate that in 2 M NaCl the inner histones are a weakly associating system with contributions from species ranging in molecular weight from dimer to octamer. The appearance of a single boundary (3.8S at 2 M NaCl) in sedimentation velocity studies conducted over a wide range of protein concentrations and ionic conditions indicates that the various histone oligomers present are in rapid equilibrium with one another. At higher salts the equilibrium is shifted to favor higher molecular weight species; in 4 M NaCl essentially all of the histone is octameric at protein concentrations above 0.2 mg/ml. The facile interconversion of histone oligomers suggests that small alterations in histone-histone interactions may be responsible for changes in nucleosome conformations during various biological processes.     

Metrizamide in D2O — A novel solute for the separation of labeled and unlabeled proteins by equilibrium density gradient centrifugation

Metrizamide(2-(3-acetamido-5-N-methylacetamido-2,4,6-triiodobenzamido)-2-deoxy-d-glucose) dissolved in D₂O was found to be a very suitable medium for the separation of labeled and unlabeled proteins by equilibrium gradient sedimentation. It is nontoxic, and has little influence on the activity of enzymes. Solutions in the density range of 1.3–1.45 g cm^?3 have low viscosities. Since the spontaneous equilibrium gradient, which is dependent on the angular velocity, occurs only after a long time of centrifugation in metrizamide solutions, the equilibrium density gradient sedimentation of proteins can be performed at the highest available speed with any preformed shallow gradient. Examples for the separation of proteins of different densities are given.     

Solution physicochemical properties of bovine beta 2-microglobulin. Aggregation states

Bovine beta 2-microglobulin (beta 2-m), the light chain of the histocompatibility antigen, was isolated in crystalline form from colostrum. Previous studies from this laboratory on the solution properties of this protein suggest the existence of a time-dependent multiple aggregation phenomenon. To clarify the molecular states of beta 2-m, its solution properties were studied by ultracentrifugation and spectropolarimetry. Sedimentation equilibrium experiments at pH 5.0 (0.08 M NaCl, 0.02 M sodium phosphate) at concentrations less than 0.3 mg/ml give Mr = 11,800. From sedimentation velocity results, we conclude that bovine beta 2-m is a much more symmetrical and compact molecule than either guinea pig or human beta 2-m. At concentrations above 0.4 mg/ml under the same conditions, sedimentation equilibrium experiments show that a monomer to tetramer reversible self-association occurs. Also, the tetramerization increases with decreasing temperature. beta 2-Microglobulin undergoes an irreversible temperature-dependent association to a much larger aggregate over a period of 7 days, as evidenced by sedimentation equilibrium and velocity results. The rate of this aggregation decreases as the pH approaches the isoelectric point (pH 7) from either side. Furthermore, circular dichroism measured at pH 5.0 under time-dependent aggregating conditions showed a marked increase in the percentage of disordered structure, leading to the conclusion that this effect is a denaturation phenomenon.     

Pressure-induced dissociation of aggregates of myelin proteolipid protein

Sedimentation velocity and equilibrium experiments have revealed an extremely pressure-sensitive aggregation of myelin proteolipid protein in the presence of Triton X-100, dissociation of the protein aggregate being observed at pressures that are several orders of magnitude lower than those effecting disaggregation of many other proteins. These results highlight the need to employ a range of angular velocities in sedimentation studies of intrinsic membrane protein.     

A strategy for efficient characterization of macromolecular heteroassociations via measurement of sedimentation equilibrium.

A method is proposed for the selection of experimental conditions for sedimentation equilibrium experiments that will provide maximal information about the values of equilibrium association constants within a given scheme for heteroassociation of two solute components. A discriminator function is proposed that indicates the sensitivity of the experimentally observed gradient or gradients to alterations in the underlying association constants. The value of this function is plotted or tabulated as a function of the concentrations of the two components, over a broad range of solution compositions. It is suggested that experiments performed with loading compositions corresponding to large absolute values of the discriminator function will yield the most information with respect to determination of the underlying association constants. This method was tested by predicting optimal conditions for three different types of sedimentation equilibrium experiments: (i) measurement of total (natural) solute absorbance; (ii) measurement of individual component gradients via measurement of tracer absorbance; and (iii) global analysis of multiple experiments. Experimental data resulting from sedimentation equilibrium experiments carried out under the specified conditions were simulated by addition of realistic levels of random error to calculated equilibrium gradients. The simulated data were then analyzed exactly as real experimental data, i.e., without prior knowledge of the underlying association constants. It was found that the highest accuracy and precision in determination of heteroassociation constants are obtained by global analysis of multiple experiments performed using significantly different loading compositions, each of which is selected from 'sensitive' regions of the discriminator map.     

The determination of density and molecular weight distributions of lipoproteins by sedimentation equilibrium.

A method is presented by which an experimental record of total concentration as a function of radial distance, obtained in a sedimentation equilibrium experiment conducted with a noninteracting mixture in the absence of a density gradient, may be analyzed to obtain the unimodal distributions of molecular weight and of partial molar volume when these vary concomitantly and continuously. Particular attention is given to the caracterization of classes of lipoproteins exhibiting Gaussian distributions of these quantities, although the analysis is applicable to other types of unimodal distribution. Equations are also formulated permitting the definition of the corresponding distributions of partial specific volume and of density. The analysis procedure is based on a method (employing Laplace transforms) developed previously, but differs from it in that it avoids the necessity of differentiating experimental results, which introduces error. The method offers certain advantages over other procedures used to characterize and compare lipoprotein samples (exhibiting unimodal distributions) with regard to the duration of the experiment, economy of the sample, and, particularly, the ability to define in principle all of the relevant distributions from one sedimentation equilibrium experiment and an external measurement of the weight average partial specific volume. These points and the steps in the analysis procedure are illustrated with experimental results obtained in the sedimentation equilibrium of a sample of human serum low density lipoprotein. The experimental parameters (such as solution density, column height, and angular velocity) used in the conduction of these experiments were selected on the basis of computer-simulated examples, which are also presented. These provide a guide for other workers interested in characterizing lipoproteins of this class.     

Analytical ultracentrifugation for the study of protein association and assembly

Analytical ultracentrifugation remains pre-eminent among the methods used to study the interactions of macromolecules under physiological conditions. Recent developments in analytical procedures allow the high resolving power of sedimentation velocity methods to be coupled to sedimentation equilibrium approaches and applied to both static and dynamic associations. Improvements in global modeling based on numerical solutions of the Lamm equation have generated new sedimentation velocity applications with an emphasis on data interpretation using sedimentation coefficient or molar mass distributions. Procedures based on the use of multiple optical signals from absorption and interference optics for the analysis of the sedimentation velocity and equilibrium behavior of more complex interactions have now been developed. New applications of tracer sedimentation equilibrium experiments and the development of a fluorescence optical system for the analytical ultracentrifuge extend the accessible concentration range over several orders of magnitude and, coupled with the new analytical procedures, provide powerful new tools for studies of both weak and strong macromolecular interactions in solution.     

Quantitative characterization of reversible molecular associations via analytical centrifugation

The ultracentrifuge provides several techniques for the quantitative characterization of reversible small molecule-macromolecule and macromolecule-macromolecule interactions in solution. The nature of the association to be studied determines the preferred technique. High speed centrifugation is the method of choice for characterizing reversible heteroassociations between species of greatly different mass (i.e., sedimentation coefficient). This technique provides a relatively rapid, artifact-free, and thermodynamically rigorous means of quantifying the amount of nonsedimenting or slowly sedimenting free ligand in equilibrium with rapidly sedimenting acceptor-bound ligand at one particular solution composition. Results obtained over a broad range of ligand and/or acceptor concentrations lead to model-independent binding isotherms that may subsequently be analyzed in the context of models for ligand-acceptor association. Lower speed centrifugation to sedimentation equilibrium is the method of choice for characterizing reversible selfassociations and for characterizing heteroassociations between components that cannot be well separated on the basis of sedimentation velocity. In the dilute limit, this technique can provide model-free information about the dependence of weight-average molecular weight of each component upon solution composition, which can subsequently be analyzed in the context of equilibrium models for self- or heteroassociation. At higher concentrations, the models must be generalized to allow for the effect of nonspecific (nonideal) interactions upon sedimentation and association. The use of tracers provides a means for greatly extending the range of solute concentrations and solution compositions over which both types of measurements may be applied, providing enhanced ability to discriminate between alternative proposed mechanisms for self- or heteroassociations.     

Partial specific volume changes of proteins: ultracentrifugal and viscometric studies

The apparent partial specific volume φ of proteins in multicomponent solutions may exhibit marked differences from those of proteins in simple two-component systems. Therefore, explicit knowledge of φ is of great importance in connection with a wide variety of biochemical techniques, especially those which involve conditions causing protein denaturation or addition of high amounts of third components (e.g. electrolytes). In the case of denaturation experiments, changes in φ are found to parallel changes in viscosity, absorbance and other molecular parameters. Attempts to correlate densimetric observations with hydrodynamic measurements show that sedimentation velocity and sedimentation equilibrium experiments in multicomponent solutions yield the appropriate results only if experimentally determined φ values are applied. However, ultracentrifugal analysis can be used to investigate changes of φ even under conditions where direct determination is not feasible, e.g. in the microgram range of protein concentration.     

Effect of temperature on the self-assembly of the Escherichia coli ClpA molecular chaperone

Protein quality control pathways rely upon ATP-dependent proteases, such as Escherichia coli ClpAP, to perform maintenance roles in the cytoplasm of the cell. ATP-dependent proteases remove misfolded and partially synthesized proteins. This action is particularly important in situations where an unregulated accumulation of such proteins will have a deleterious effect on the cell. ClpAP is composed of a tetradecameric serine protease, ClpP (21.6 kDa monomer), and the ATPase/protein unfoldase ClpA (84.2 kDa monomer). ClpA also uses its protein unfolding activity to remodel proteins and protein complexes; thus, in the absence of the proteolytic component, ClpA is considered a molecular chaperone. Previous reports, by others, suggested that ClpA exists in a monomer-dimer equilibrium at 4 °C. In contrast, using a combination of sedimentation velocity, sedimentation equilibrium, and dynamic light scattering, we recently reported that ClpA exists in a monomer-tetramer equilibrium at 25 °C. Here we report an investigation of the effect of temperature on the self-association of the E. coli ClpA protein unfoldase using analytical ultracentrifugation techniques. The results of sedimentation velocity and sedimentation equilibrium experiments performed at multiple loading concentrations of ClpA over a range of temperatures from 3.9 to 38.2 °C are discussed. Sedimentation velocity experiments show a decrease in weight average s(20,w) at the extremes of temperature. This result, along with extensive sedimentation equilibrium data and analysis, suggests the presence of a dimeric intermediate of ClpA that is differentially populated as a function of temperature. Further, analysis of sedimentation equilibrium data as a function of temperature led us to propose a monomer-dimer-tetramer equilibrium to describe the temperature dependence of ClpA self-assembly in the absence of nucleotide.     

The isolation of aggregates of spectrin from bovine erythrocyte membranes.

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4% agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960 000 and 480 000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physical properties of spectrin.     

Caldesmon. Molecular weight and subunit composition by analytical ultracentrifugation

A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. There have also been conflicting reports concerning whether caldesmon is a monomer or dimer. We attempted to resolve these uncertainties by determining the molecular weight of chicken gizzard smooth muscle caldesmon using the technique of sedimentation equilibrium in the analytical ultracentrifuge. Unlike previous methods that have been used to estimate the molecular weight of caldesmon, the molecular weight determined by equilibrium sedimentation does not depend upon assumptions about the shape of the molecule. We concluded that caldesmon in solution is monomeric with a molecular mass of 93 +/- 4 kDa, a value that is much less than those previously reported in the literature. This new value, in conjunction with sedimentation velocity experiments, led to the conclusion that caldesmon is a highly asymmetric molecule with an apparent length of 740 A in solution. The mass of a cyanogen bromide fragment, with an apparent mass of 37 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was determined to be 25.1 +/- 0.6 kDa using sedimentation equilibrium. These results imply that the reported molecular weights of other fragment(s) of caldesmon have also been overestimated. We have determined an optical extinction coefficient for caldesmon (E1%(280 nm) = 3.3) by determining its concentration from its refractive index which was measured in the analytical ultracentrifuge. From the above values of the molecular weight and the extinction coefficient, we redetermined that the caldesmon molecule has two cysteines and recalculated the stoichiometric molar ratio of actin/tropomyosin/caldesmon in the smooth muscle thin filament to be 28:4:1.     

Self-association of sperm whale metmyoglobin

The solution behavior of sperm whale metmyoglobin in 0.15 I phosphate-chloride buffer, pH 7.2, has been examined by sedimentation equilibrium, frontal gel chromatography, and sedimentation velocity. Results obtained from all three studies are shown to be consistent with a self-association model in which dimerization of the myoglobin is governed by an association equilibrium constant of 0.068 liter/g (580 M-1) at 20 degrees C.     

Motion detection and adaptation in crayfish photoreceptors. A spatiotemporal analysis of linear movement sensitivity

Impulse and sine wave responses of crayfish photoreceptors were examined to establish the limits and the parameters of linear behavior. These receptors exhibit simple low pass behavior which is well described by the transfer function of a linear resistor-capacitor cascade of three to five stages, each with the same time constant (tau). Additionally, variations in mean light intensity modify tau twofold and the contrast sensitivity by fourfold. The angular sensitivity profile is Gaussian and the acceptance angle (phi) increases 3.2-fold with dark adaptation. The responses to moving stripes of positive and negative contrast were measured over a 100-fold velocity range. The amplitude, phase, and waveform of these responses were predicted from the convolution of the receptor's impulse response and angular sensitivity profile. A theoretical calculation based on the convolution of a linear impulse response and a Gaussian sensitivity profile indicates that the sensitivity to variations in stimulus velocity is determined by the ratio phi/tau. These two parameters are sufficient to predict the velocity of the half-maximal response over a wide range of ambient illumination levels. Because phi and tau vary in parallel during light adaptation, it is inferred that many arthropods can maintain approximately constant velocity sensitivity during large shifts in mean illumination and receptor time constant. The results are discussed relative to other arthropod and vertebrate receptors and the strategies that have evolved for movement detection in varying ambient illumination.     

Effects of pressure on the composition density distribution in the ultracentrifuge

The effects of pressure on the composition density distribution in a binary density gradient at sedimentation equilibrium in the analytical ultracentrifuge are rigorously examined. A computer algorithm is described for the necessary iterative computations. The pressure effect is found to be significant in runs where a long column length, high angular velocity, and a salt with a high pressure correction coefficient are simultaneously employed. Such conditions are sometimes encountered in current studies in which high precision is required to measure the compressibility of proteins.     

Convection-free boundary sedimentation of dilute protein solutions

Use of the density gradient sedimentation velocity technique appears to be essential for the accurate determination of the mean sedimentation coefficients of dilute protein solutions. When performed on an analytical ultracentrifuge equipped with a photoelectric-scanning-absorption optical system, the density gradient sedimentation velocity technique has been shown to be particularly useful in studying the subunit association-dissociation equilibria of multisubunit enzyme systems. The time factor has been shown to be a major advantage of the density gradient sedimentation velocity technique, as opposed to the sedimentation equilibrium technique, in studying the subunit association-dissociation equilibria of multisubunit enzymes such as rabbit muscle apo-glyceraldehyde-3-phosphate dehydrogenase, which is very unstable in dilute solution.     

Sedimentation velocity, multi-speed method for analyzing polydisperse solutions

A method is described for the sedimentation velocity analysis of solutions composed of macromolecular solutes of widely disparate size. In sedimentation velocity experiments, usually a single rotor speed is chosen for the entire run, and consequently, the range of observable sedimentation coefficients can be severely limited. This limitation can be removed if the speed is varied during the run, starting with a relatively low speed so that the largest particles can be easily observed. The speed is increased during the run until full speed is attained and the run continued at full speed until the smallest species of interest have cleared the solution. The method, called wide distribution analysis, is based on the method developed originally by Yphantis and co-workers (Proc. Natl. Acad. Sci. USA 78 (1981) 1431) and on the time derivative method of Stafford (Anal. Biochem. 203 (1992) 295), essentially eliminating both the time-independent and radially-independent noise thereby improving the precision, especially for interference optics. An algorithm for analysis of data from both absorbance and interference optics and experimental protocols compatible with the Beckman XL-I Analytical Ultracentrifuge are presented. With these protocols an extremely wide range of sedimentation coefficients from approximately 1.0 to 250000 S can be accommodated in a single multi-speed run.     

Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability

The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 --> Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.     

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.     

Effect of protein concentration on the molecular weight of delta5-3-ketosteroid isomerase.

The molecular weight of delta-5-3-ketosteroid isomerase from Pseudomonas testosteroni was determined by means of sedimentation equilibrium and exclusion chromatography over a wide range of enzyme concentrations in 0.2 M potassium phosphate buffer, pH 7.0. In addition, the sedimentation constant of the enzyme was determinded over an extended range of concentrations. The enzyme was found to have a molecular weight of 26,000 plus or equal to 1,000, suggesting that it is a dimer of identical or similar 13,400 molecular weight polypeptide chains. In the ultracentrifuge this dimeric species was found to undergo aggregation at enzyme concentrations above 2 mg per ml and dissociation at enzyme concentrations below 0.05 mg per ml. Exclusion chromatography studies indicate that under the conditions of chromatography the oligomeric enzyme is partially dissociated at enzyme concentrations in the range 0.2 to 0.002 mug per ml. These results suggest that under conditions of enzyme assay in 0.2 M potassium phosphate buffer, pH 7.0, isomerase is in a monomeric state of aggregation.