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.     

Adair was right in his time

The subunit molar mass of hemoglobin was established in the 19th century by chemical analysis, the tetramer structure by osmotic pressure determination in 1924 and by the newly developed analytical ultracentrifuge in 1926, which became a powerful tool for biological macromolecule molar mass determinations. The Svedberg equation was derived by eliminating the translational friction coefficient relating to sedimentation and diffusion in the ultracentrifuge in a strictly solute/solvent vanishing concentration two-component system analysis. A differential equation describing the radial equilibrium concentration distribution in the ultracentrifuge was also derived, both yielding the buoyant molar mass (1-nu2rho)M2 term. Many years later it was realized that solutions of biological macromolecules are multicomponent systems and the two-component analysis leads to minor or major erroneous results. Thermodynamic derivation of an equation for multicomponent systems redefines the buoyant molar mass terms by (deltarho/deltac2)muM2, leading to correct molar mass (g/mol) values following determination of the density increment at constant chemical potentials of diffusible solutes, and powerfully connects the analytical sedimentation equation to the osmotic pressure concentration derivative and, in a broad complementary sense, to light, X-ray and neutron scattering experiments. Macromolecular interactions can be studied with high precision and solute-solvent interactions yield powerful information relating to "thermodynamic" hydration, closely related to hydration derived from X-ray diffraction, as well as solute-cosolute interactions. A series of examples is given to demonstrate the correctness and usefulness of the thermodynamic multicomponent system approach. It is a strange fact that in current analytical ultracentrifugation analysis the elegant and powerful multicomponent solution technology is almost totally disregarded and the classical limited validity Svedberg approach is used uniquely.     

Zone Centrifugation in a Cesium Chloride Density Gradient Caused by Temperature Change

In this communication is described a new technique for the determination of sedimentation coefficients of macromolecules banded in equilibrium density gradients. Initially, the macromolecules are banded in the analytical ultracentrifuge at a low temperature of about 5°C. After equilibrium has been obtained, the temperature is increased to 25°C. The equilibrium band will now sediment to a new equilibrium position in the ultracentrifuge cell: (a) By following the position of the migrating band as a function of time, sedimentation coefficients may be determined. (b) If several species having different sedimentation coefficients are present in the original band, then during the course of the migration the band may split into several new bands which eventually reunite at the final equilibrium position. (c) If different chemical species of macromolecules such as nucleic acids and carbohydrates are present, in general they will exhibit different temperature density relationships, and can move different distances and directions in response to temperature change.     

A parameterized overspeeding method for the rapid attainment of low-speed sedimentation equilibrium

The approach of a solution of dilute, monodisperse, globular macromolecules to low-speed sedimentation equilibrium in an ultracentrifuge is simulated by numerical integration of the Lamm equation. Various combinations of overspeed time and angular velocity are used to assess the conditions needed to minimize the time it takes the solution to attain sedimentation equilibrium. The optimal overspeeding time and angular velocity are determined over a wide range of values of the molecular weight (relative molar mass) of the solute and the radial distance between the meniscus and base of the solution. The results may be expressed as simple functions which allow facile calculation of (a) the optimal overspeeding time and velocity, and (b) the time required to reach sedimentation equilibrium. The results are in reasonable agreement with previous analytical solutions which were based on several simplifying assumptions. The parameterized overspeeding procedure is shown to be robust over a wide range of conditions, and typically leads to a greater than 5-fold reduction in centrifugation time.     

Characterization of heterologous protein-protein interactions using analytical ultracentrifugation.

Methods for quantitative characterization of heterologous protein-protein interactions by means of analytical ultracentrifugation (AUC) include sedimentation equilibrium, tracer sedimentation equilibrium, sedimentation velocity, and analytical band sedimentation. Fundamental principles governing the behavior of macromolecules in a centrifugal field are summarized, and the application of these principles to the interpretation of data obtained from each type of experiment is reviewed. Instrumentation and software for the acquisition and analysis of data obtained from different types of AUC experiments are described.     

An automated method for determination of the sedimentation coefficient of macromolecules using a preparative centrifuge

Following centrifugation in a preparative ultracentrifuge at relatively high speeds for 1-3 h, quartz tubes containing approximately 150 microliter of macromolecular solution are optically scanned using an apparatus and method previously described (Attri, A. K., and Minton A. P. (1983) Anal. Biochem. 133, 142-152). The resulting data are processed by microcomputer immediately upon completion of scanning to yield the sedimentation coefficient of the solute. Calculated values agree to within a few percent with those found in the literature and with the results of control experiments carried out using an analytical ultracentrifuge.     

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.     

Biophysical studies by ultracentrifugation.

Advances in data analysis are broadening the applicability of ultracentrifugation to the characterization of macromolecular behavior in complex solution. The direct fitting of sedimentation velocity data to the Lamm equation is emerging as a very powerful means to characterize size distributions, improve the precision of data analysis and increase experimental throughput. With improvements in data acquisition and analysis, ultracentrifugation is poised to make significant contributions to our understanding of how macromolecules behave in vivo.     

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.     

Advances in sedimentation velocity analysis.

On February 20, 1996, a workshop titled "Advances in Sedimentation Velocity Analysis" was held at the Biophysical Society meeting in Baltimore, Maryland, in honor of Professor David Yphantis's 65th birthday. Although he is known more for his work with sedimentation equilibrium, David's work on instrumentation and data analysis is the foundation for many of the recent advances in both equilibrium and velocity sedimentation. Over the years he has trained numerous graduate students, most of whom have gone on to emphasize the use of analytical ultracentrifugation to answer biochemical questions involving macromolecular assembly. His laboratory was one of very few that continued to use and develop analytical ultracentrifugation during its nadir in the 1970s and early 1980s. The rebirth and resurgence of analytical ultracentrifugation owe a great deal to his persistence and enthusiasm. These efforts have borne fruit. In the last five years, through his work at the National Analytical Ultracentrifugation Facility, he has helped train nearly 100 individuals in the delicate art of nonlinear least-squares analysis of equilibrium sedimentation data. Furthermore, the number of researchers using the ultracentrifuge and the number of papers published has skyrocketed in the last few years. This workshop, then, was a way to thank David for his years of devotion to analytical ultracentrifugation.     

An asymmetric and slightly dimerized structure for the tetanus toxoid protein used in glycoconjugate vaccines

Tetanus toxoid protein has been characterized with regard oligomeric state and hydrodynamic (low-resolution) shape, important parameters with regard its use in glycoconjugate vaccines. From sedimentation velocity and sedimentation equilibrium analysis in the analytical ultracentrifuge tetanus toxoid protein is shown to be mostly monomeric in solution (～86%) with approximately 14% dimer. The relative proportions do not appear to change significantly with concentration, suggesting the two components are not in reversible equilibrium. Hydrodynamic solution conformation studies based on high precision viscometry, combined with sedimentation data show the protein to be slightly extended conformation in solution with an aspect ratio ～3. The asymmetric structure presents a greater surface area for conjugation with polysaccharide than a more globular structure, underpinning its popular choice as a conjugation protein for glycoconjugate vaccines.     

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.     

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.     

Studies on polydisperse systems using an air-driven ultracentrifuge: application to phosphatidylcholine vesicles.

A high-speed air-driven ultracentrifuge (Airfuge) has been used to determine the molecular weight and effective specific volume of phosphatidylcholine vesicles. The method used to determine the effective specific volume involved varying the solution density until zero sedimentation of the vesicles occurred. The value obtained for the effective specific volume of 0.9885 ml/g agrees well with previously reported values. The determination of the molecular weight of the vesicles is based on a method in which the fraction of vesicles remaining in an upper fraction of the solution column is compared with the values obtained using standard proteins. The values obtained for the molecular weight of the vesicles range from 1.7 X 10(6) to 2.3 X 10(6) and are in good agreement with results obtained using the analytical ultracentrifuge and with previously reported results. Possible effects due to the polydispersity of the solute are assessed using theoretical calculations and the possibility of using the Airfuge for the study of other polydisperse systems is discussed.     

Earl Sutherland (1915-1975) and the discovery of cyclic AMP

In 1945, Earl Sutherland (1915-1975) and associates began studies of the mechanism of hormone-induced glycogen breakdown in the liver. In 1956, their efforts culminated in the identification of cyclic AMP, an ancient molecule generated in many cell types in response to hormonal and other extracellular signals. Cyclic AMP, the original "second messenger," transmits such signals through pathways that regulate a diversity of cellular functions and capabilities: metabolic processes such as lipolysis and glycogenolysis; hormone secretion; the permeability of ion channels; gene expression; cell proliferation and survival. Indeed, it can be argued that the discovery of cyclic AMP initiated the study of intracellular signaling pathways, a major focus of contemporary biomedical inquiry. This review presents relevant details of Sutherland's career; summarizes key contributions of his mentors, Carl and Gerti Cori, to the knowledge of glycogen metabolism (contributions that were the foundation for his own research); describes the experiments that led to his identification, isolation, and characterization of cyclic AMP; assesses the significance of his work; and considers some aspects of the impact of cyclic nucleotide research on clinical medicine.     

Fluorescence detection for the XLI analytical ultracentrifuge

Analytical ultracentrifugation (AUC) provides first-principle hydrodynamic and thermodynamic information concerning the size, shape and interactions of macromolecules. The fundamental measurement needed in AUC is the macromolecular concentration as a function of radial position and time. Currently, the Beckman Coulter XLI analytical ultracentrifuge may be equipped with absorbance and refractive detectors, which provide complementary concentration determinations. For detecting trace quantities of materials, fluorescence detection offers unique advantages over either absorbance or interference detection. A prototype fluorescence detector for the XLI analytical ultracentrifuge has been developed and its characteristics determined. An Ar(+) laser provides a continuous 488-nm excitation beam. Radial resolution is achieved by scanning the focused beam along a radial axis. Detection of the fluorescence signal uses a co-axial, front-face optical configuration to reduce inaccuracies in the concentration caused by inner filter effects. A high-speed A/D data acquisition system allows the fluorescence intensity to be monitored continuously and at a sufficiently high angular resolution so that at any radial position the intensities from all of the samples may be acquired at each revolution. The fluorescence detector is capable of detecting concentrations as low as 300 pM for fluorescein-like labels. The radial resolution of the fluorescence detector is comparable to that of the absorbance system. Both sedimentation velocity and sedimentation equilibrium measurements may be made with the fluorescence detector. Results are presented comparing data acquired using the fluorescence with those acquired using the absorbance detector.     

An automated method for determining buoyant density of nucleic acids using a preparative ultracentrifuge

We present a technique for analytical buoyant density sedimentation of nucleic acids which is performed in a preparative ultracentrifuge, in contrast to an analytical ultracentrifuge. Following centrifugation in a preparative rotor, small cylindrical quartz tubes are optically scanned; upon completion of the scan the data are processed immediately by a microcomputer and the buoyant density of the nucleic acid is calculated. Experimental data are presented employing several different deoxyribonucleic acids banded in neutral and alkaline cesium sulfate. Results are independent of rotor speed, location of bands within the gradient, and loading density of the cesium sulfate solution. Derived buoyant density values agree within 0.5% of previously published values.     

Estimating friction coefficients of mixed globular/chain molecules, such as protein/DNA complexes.

Existing methods for predicting translational friction properties of complex molecules start by explicitly building up their three-dimensional shape with spherical subunits. This treatment has been used especially for two types of systems: rigid assemblies and flexible chain molecules. However, many protein/DNA complexes such as chromatin consist of a small number of globular, relatively rigid, bound protein interspersed by long stretches of flexible DNA chain. I present a higher level of treatment of such macromolecules that avoids explicit subunit modeling as much as possible. An existing analytical formulation of the hydrodynamics equations is shown to be accurate when used with the present treatment. Thus the approach is fast and can be applied to hydrodynamic studies of highly degenerate multiple equilibria, such as those encountered in problems of the regulation of chromatin structure. I demonstrate the approach by predicting the effect of a hypothetical unwinding process in dinucleosomes and by simulating the distribution of sedimentation coefficients for cooperative and random models for a chromatin saturation process.     

Accounting for thermodynamic non-ideality in the Guinier region of small-angle scattering data of proteins

Hydrodynamic studies of the solution properties of proteins and other biological macromolecules are often hard to interpret when the sample is present at a reasonably concentrated solution. The reason for this is that solutions exhibit deviations from ideal behaviour which is manifested as thermodynamic non-ideality. The range of concentrations at which this behaviour typically is exhibited is as low as 1–2 mg/ml, well within the range of concentrations used for their analysis by techniques such as small-angle scattering. Here we discuss thermodynamic non-ideality used previously used in the context of light scattering and sedimentation equilibrium analytical ultracentrifugation and apply it to the Guinier region of small-angle scattering data. The results show that there is a complementarity between the radially averaged structure factor derived from small-angle X-ray scattering/small-angle neutron scattering studies and the second virial coefficient derived from sedimentation equilibrium analytical ultracentrifugation experiments.