Sedimentation Equilibrium Analytical Ultracentrifugation Research Articles

Experimental Method to Distinguish between a Solution and a Suspension

AbstractDispersion of objects in a fluid phase can be classified as solutions (Gibbs free energy of mixing, ΔGmix < 0) or suspensions (ΔGmix > 0) depending on their thermodynamic stability. Small objects tend to form solutions, larger ones suspensions, e.g., molecules versus micrometer‐sized colloids. Proteins and nanomaterials fall between these two size regimes. The long‐standing issue of whether proteins and nanoparticles are dissolved or suspended remains an important research question. Here, a simple, versatile, and experimentally robust method, based on sedimentation equilibrium analytical ultracentrifugation (SE‐AUC), which can determine whether proteins, nanoparticles, or polymers form solutions or suspensions, is presented. SE‐AUC determines the osmotic pressure profile for a dispersion. Such a profile for solutions (equilibrium one‐phase systems) is independent of the initial and the operating conditions. The opposite is true for suspensions that are nonequilibrium two‐phase systems. This study proves that bovine serum albumin and lysozyme form solutions while ferritin and apoferritin form suspensions.

Analyzing the weak dimerization of a cellulose binding module by analytical ultracentrifugation

Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBMCipA from Clostridium thermocellum. Here we studied the oligomerization behavior of CBMCipA, as such solution association may have an impact on its use. As the principal approach, we used sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data by a discrete model genetic algorithm and by building a binding isotherm based on weight average sedimentation coefficient and by global fitting of sedimentation equilibrium data we found that the CBMCipA shows a weak dimerization interaction with a dissociation constant KD of 90 ± 30 μM. As the KD of CBMCipA binding to cellulose is below 1 μM, we conclude that the dimerization is unlikely to affect cellulose binding. However, at high concentrations used in some applications of the CBMCipA, its dimerization is likely to have a marked effect on its solution behavior.

Polysaccharide valproates: Structure - property relationships in solution

Polysaccharides are promising macromolecular platforms for use in the life sciences. Here, bioactive cellulose, pullulan, and dextran valproates are characterized hydrodynamically by sedimentation velocity and thermodynamically by sedimentation equilibrium analytical ultracentrifugation. Using sedimentation-diffusion analysis of sedimentation velocity experiments by numerical solution of the Lamm equation enabled the calculation of sedimentation and diffusion coefficients, and consequently molar masses. Sedimentation equilibrium experiments were then also used to determine the average molar masses. The corresponding set of data, with independently performed self-diffusion measurements by nuclear magnetic resonance spectroscopy, and together with size exclusion chromatography molar masses by coupling to refractive index-, viscometric-, and multi-angle laser light scattering detection, were subsequently correlated to each other by the hydrodynamic invariant and sedimentation parameter. We assess statistically most relevant average values of the molar masses of these polysaccharide valproates with relevant macromolecular conformational characteristics.

A Reappraisal of Sedimentation Nonideality Coefficients for the Analysis of Weak Interactions of Therapeutic Proteins

The study of weak or colloidal interactions of therapeutic proteins in different formulations allows prediction and optimization of protein stability. Various biophysical techniques have been applied to determine the second osmotic virial coefficient B2 as it reflects on the macromolecular distance distribution that governs solution behavior at high concentration. In the present work, we exploit a direct link predicted by hydrodynamic theory between B2 and the nonideality of sedimentation, commonly measured in sedimentation velocity analytical ultracentrifugation through the nonideality coefficient of sedimentation, kS. Using sedimentation equilibrium analytical ultracentrifugation for independent measurement of B2, we have examined the dependence of kS on B2 for model proteins in different buffers. The data exhibit the expected linear relationship and highlight the impact of protein shape on the magnitude of the nonideality coefficient kS. Recently, measurements of kS have been considerably simplified allowing higher throughput and simultaneous polydispersity assessment at higher protein concentrations. Thus, sedimentation velocity may offer a useful approach to compare the impact of formulation conditions on weak interactions and simultaneously on higher-order structure of therapeutic proteins.

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.

Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium

Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) is a gold standard for the rigorous determination of macromolecular buoyant molar masses and the thermodynamic study of reversible interactions in solution. A significant experimental drawback is the long time required to attain SE, which is usually on the order of days. We have developed a method for time-optimized SE (toSE) with defined time-varying centrifugal fields that allow SE to be attained in a significantly (up to 10-fold) shorter time than is usually required. To achieve this, numerical Lamm equation solutions for sedimentation in time-varying fields are computed based on initial estimates of macromolecular transport properties. A parameterized rotor-speed schedule is optimized with the goal of achieving a minimal time to equilibrium while limiting transient sample preconcentration at the base of the solution column. The resulting rotor-speed schedule may include multiple over- and underspeeding phases, balancing the formation of gradients from strong sedimentation fluxes with periods of high diffusional transport. The computation is carried out in a new software program called TOSE, which also facilitates convenient experimental implementation. Further, we extend AUC data analysis to sedimentation processes in such time-varying centrifugal fields. Due to the initially high centrifugal fields in toSE and the resulting strong migration, it is possible to extract sedimentation coefficient distributions from the early data. This can provide better estimates of the size of macromolecular complexes and report on sample homogeneity early on, which may be used to further refine the prediction of the rotor-speed schedule. In this manner, the toSE experiment can be adapted in real time to the system under study, maximizing both the information content and the time efficiency of SE experiments.

Characterization of Homogeneous, Cooperative Protein-DNA Clusters by Sedimentation Equilibrium Analytical Ultracentrifugation and Atomic Force Microscopy.

Strong, positively cooperative binding can lead to the clustering of proteins on DNA. Here, we describe one approach to the analysis of such clusters. Our example is based on recent studies of the interactions of O(6)-alkylguanine DNA alkyltransferase (AGT) with high-molecular-weight DNAs (Adams et al., 2009; Tessmer, Melikishvili, & Fried, 2012). Cooperative cluster size distributions are predicted using the simplest homogeneous binding and cooperativity (HBC) model, together with data obtained by sedimentation equilibrium analysis. These predictions are tested using atomic force microscopy imaging; for AGT, measured cluster sizes are found to be significantly smaller than those predicted by the HBC model. A mechanism that may account for cluster size limitation is briefly discussed.

Characterization of Capsular Polysaccharides and Their Glycoconjugates by Hydrodynamic Methods.

Hydrodynamic methods are relevant for the characterization of carbohydrates such as capsular bacterial polysaccharides or glycoconjugates in solution. This chapter focuses on the following hydrodynamic methods: sedimentation velocity analytical ultracentrifugation (SV AUC), dynamic light scattering (DLS), sedimentation equilibrium analytical ultracentrifugation (SE AUC), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and capillary viscometry-intrinsic viscosity measurement. The chapter highlights the general principle of these five methods, describes experimental details, and specifies advances in the last years.

Sedimentation in a Time-Varying Centrifugal Field for Rapid Attainment of Sedimentation Equilibrium

Sedimentation equilibrium (SE) analytical ultracentrifugation is a gold standard for the rigorous thermodynamic study of buoyant molecular weight and reversible interactions of macromolecules in solution. A significant drawback is the long experiment time, as it takes days to attain SE with standard solution columns. We have developed a new method for using a time-varying centrifugal field optimized such as to attain SE in significantly shorter time than usually required. Experimental data show that this permits long-column SE experiments to be carried out in times comparable to sedimentation velocity experiments, approximately fivefold shorter than standard SE. In contrast to the classical initial overspeeding method, which uses a single initial speed, we employ a freely varying rotor speed profile during an initial phase, for example, parameterized as a step-wise modulated exponential decay to the desired SE rotor speed. The rotor speed schedule is computationally optimized on the basis of numerical Lamm equation solutions for given macromolecular sedimentation parameter estimates, with the goal to provide a rapid attainment of equilibrium without the drawback of strong transient sample pre-concentration at the base of the solution column. The resulting rotor speed schedule frequently includes both over- and under-speeding sequences, and can be conveniently implemented on the Optima XLA/I analytical ultracentrifuge. We extended AUC data analysis models in SEDFIT to permit the analysis of concentration profiles in arbitrarily time-varying fields, to make it possible to exploit the migration in the initially high centrifugal field for estimates on macromolecular sedimentation parameters, which may be used in real-time to refine the prediction of the rotor speed schedule, so that the SE experiment can be optimized in both information content and time efficiency.

SEDFIT-MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge.

Sedimentation equilibrium (analytical ultracentrifugation) is one of the most inherently suitable methods for the determination of average molecular weights and molecular weight distributions of polymers, because of its absolute basis (no conformation assumptions) and inherent fractionation ability (without the need for columns or membranes and associated assumptions over inertness). With modern instrumentation it is also possible to run up to 21 samples simultaneously in a single run. Its application has been severely hampered because of difficulties in terms of baseline determination (incorporating estimation of the concentration at the air/solution meniscus) and complexity of the analysis procedures. We describe a new method for baseline determination based on a smart-smoothing principle and built into the highly popular platform SEDFIT for the analysis of the sedimentation behavior of natural and synthetic polymer materials. The SEDFIT-MSTAR procedure - which takes only a few minutes to perform - is tested with four synthetic data sets (including a significantly non-ideal system), a naturally occurring protein (human IgG1) and two naturally occurring carbohydrate polymers (pullulan and λ-carrageenan) in terms of (i) weight average molecular weight for the whole distribution of species in the sample (ii) the variation in "point" average molecular weight with local concentration in the ultracentrifuge cell and (iii) molecular weight distribution.

Allosteric Regulation of the Epidermal Growth Factor Receptor

It is well established that aberrant activation of the Epidermal Growth Factor Receptor (EGFR) drives cellular transformation and oncogenesis. A series of activating mutations in the extracellular region of EGFR have been identified in glioblastoma multiforme (GBM) patients, and shown to transform NIH 3T3 cells. Sedimentation equilibrium analytical ultracentrifugation analysis demonstrates that, in the context of the soluble EGFR extracellular region (sEGFR), these mutations are monomeric in the absence of ligand and dimerize upon ligand binding with affinities that are similar to those observed for the wildtype receptor. We also find that these mutations elevate the affinity of growth factor binding to sEGFR, and that there are differential effects on the entropic driving forces of growth factor binding to sEGFR. Taken together, these results demonstrate a potentially novel mode of EGFR disregulation by these GBM mutations. Our recent detailed thermodynamic analysis of ligand binding and dimerization in the EGFR system suggests that a greater understanding of the changes that occur upon binding of ligand to EGFR will be required before we can appreciate the mechanisms of disregulation by oncogenic mutation. We will present our progress towards determining a profile of the dynamic and conformational effects of ligand binding to EGFR using associated hydrogen/deuterium exchange coupled to mass spectrometry (HX/MS). Comparing these profiles to those for EGFR bearing oncogenic mutations may reveal why these single amino acid substitutions can lead to receptor activation. Progress towards the application of these techniques to near full length receptors reconstituted in a lipid nanodisc model membrane system will also be reported, which will provide valuable information about the structurally poorly characterized juxtamembrane regions of EGFR.

Effect of Pegylation on Self-Association of IFN-α2b

Pegylation of therapeutic proteins is an established technology used to enhance the bioavailability of an active pharmaceutical ingredient in the body of patients. While the physiochemical properties of pegylated monomeric proteins have been extensively described, there is still limited information on the characterization of pegylated oligomeric proteins. In this study, we report the characterization of a pegylated interferon alpha2b (PEGIFN-α2b) concentration-dependent oligomerization by a series of orthogonal biochemical and biophysical methods. These methods include sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, matrix-assisted laser desorption ionization, and size exclusion chromatography of bissulfosuccinimidyl suberate cross-linked PEGIFN. We report here that PEGIFN-α2b self-associates in a concentration-dependent manner into mainly monomers, dimers, and trimers. In the presence of the chemical cross-linker, PEGIFN-α2b is primarily monomeric (57%) at concentration lower than 0.3 mg/mL and contains about equal amount of monomers and dimers (47.0% and 37.7%, respectively), about 15% of trimers, and up to 4% of higher molecular weight species at 0.7 mg/mL and above.

Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation

AbstractModern computational strategies have allowed for the direct modeling of the sedimentation process of heterogeneous mixtures, resulting in sedimentation velocity (SV) size‐distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of analytical ultracentrifugation (AUC), such as the study of protein self‐ and hetero‐association, the study of membrane proteins, and applications in biotechnology. New global multisignal modeling and mass conservation approaches in SV and sedimentation equilibrium (SE), in conjunction with the effective‐particle framework for interpreting the sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multiprotein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current unit is to describe the current tools for the study of soluble proteins, detergent‐solubilized membrane proteins and their interactions by SV and SE. Curr. Protoc. Protein Sci. 71:20.12.1‐20.12.49. © 2013 by John Wiley & Sons, Inc.

Pathogenic Serum Amyloid A 1.1 Shows a Long Oligomer-rich Fibrillation Lag Phase Contrary to the Highly Amyloidogenic Non-pathogenic SAA2.2

Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

Structure and dimerization of translation initiation factor aIF5B in solution

Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0–6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s 20 , w 0 were determined to be 3.64 and 5.51 ± 0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. [6]) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p( r) function indicated an elongated conformation supported by a radius of gyration of 37.5 ± 0.2 Å and a maximum dimension of ∼130 Å. The effects of glycerol on the formation of dimers are discussed. This new model of aIF5B in solution shows that there are universal structural differences between aIF5B and the homologous protein IF2 from Escherichia coli.

Evaluation of sugammadex self-association

Sugammadex, a thiolated γCD derivative used as an antagonist of steroidal blockers, was studied with regard to its tendency to self-associate in aqueous solution. Three independent methods – permeation through semi-permeable cellophane membranes, dynamic light scattering, and sedimentation equilibrium analytical ultracentrifugation – were used for this purpose. The results were in agreement with each other and showed no evidence of self-association in a wide sugammadex concentration range from 0.25 to 100 mg/ml.

Self-association studies of the bifunctional N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli.

The N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a key bifunctional enzyme in the biosynthesis of UDP-GlcNAc, a precursor in the synthesis of cell wall peptidoglycan. Crystal structures of the enzyme from different bacterial strains showed that the polypeptide forms a trimer through a unique parallel left-handed beta helix domain. Here, we show that the GlmU enzyme from Escherichia coli forms a hexamer in solution. Sedimentation equilibrium analytical ultracentrifugation demonstrated that the enzyme is in a trimer/hexamer equilibrium. Small-angle X-ray scattering studies were performed to determine the structure of the hexameric assembly and showed that two trimers assemble through their N-terminal domains. The interaction is mediated by a loop that undergoes a large conformational change in the uridyl transferase reaction, a feature that may affect the enzymatic activity of GlmU.

Chaperone-like N-Methyl Peptide Inhibitors of Polyglutamine Aggregation

Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into beta-sheet-rich insoluble aggregates associated with Huntington's disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe(2) [Anth-K-Q-Q(Me(2))-Q-Q(Me(2))-Q-CONH(2), where Anth is N-methylanthranilic acid and Q(Me(2)) is side chain N-methyl Q], has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe(2) can completely prevent fibrillation of a synthetic aggregating peptide, YAQ(12)A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K(d) with very fast k(on) and k(off) values. Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe(2) is predominantly or entirely monomeric at concentrations of <or=1 mM and that it forms a 1:1 stoichiometric complex with a fibril-forming target, YAQ(12)A. 5QMe(2) inhibits not only nucleation of YAQ(12)A but also fibril extension, as shown by the fact that it also inhibits seeded fibril growth where the nucleation steps are bypassed. 5QMe(2) acts on its targets only when they are in the PPII-like conformation, but not after they undergo a transition to beta-sheets. Thus, 5QMe(2) does not disassemble preformed YAQ(12)A; this contrasts with our previously described, backbone N-methylated inhibitors of beta-amyloid aggregation [Gordon, D. J., et al. (2001) Biochemistry 40, 8237-8245; Gordon, D. J., et al. (2002) J. Pept. Res. 60, 37-55]. The mode of action of 5QMe(2) is reminiscent of that of chaperones, because it binds and releases its targets very rapidly and maintains them in a nonaggregation-prone, monomeric state, in this case, the polyproline II (PPII)-like conformation, as shown by circular dichroism spectroscopy.

Determination of protein complex stoichiometry through multisignal sedimentation velocity experiments

Determination of the stoichiometry of macromolecular assemblies is fundamental to an understanding of how they function. Many different biophysical methodologies may be used to determine stoichiometry. In the past, both sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation have been employed to determine component stoichiometries. Recently, a method of globally analyzing multisignal sedimentation velocity data was introduced by Schuck and coworkers. This global analysis removes some of the experimental inconveniences and inaccuracies that could occur in the previously used strategies. This method uses spectral differences between the macromolecular components to decompose the well-known c( s) distribution into component distributions c k ( s); that is, each component k has its own c k ( s) distribution. Integration of these distributions allows the calculation of the populations of each component in cosedimenting complexes, yielding their stoichiometry. In our laboratories, we have used this method extensively to determine the component stoichiometries of several protein–protein complexes involved in cytoskeletal remodeling, sugar metabolism, and host–pathogen interactions. The overall method is described in detail in this work, as are experimental examples and caveats.

Solution structure of the human signaling protein RACK1

BackgroundThe adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development.ResultsIn this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion.ConclusionOur SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.