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Page 1. EMERGING BIOCHEMICAL AND BIOPHYSICAL TECHNIQUE S Series Editor: Todd M. Schuster MODERN ANALYTICAL ULTRACENTRIFUGATION Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems TODD M . SCHUSTER ...
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Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate means for characterizing solution hydrodynamic and thermodynamic properties. In recent times AUC has found broad application in the... more
Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate means for characterizing solution hydrodynamic and thermodynamic properties. In recent times AUC has found broad application in the biopharmaceutical industry as a first-principle means for quantitatively characterizing biopharmaceuticals. Boundary sedimentation velocity AUC (SV-AUC) analysis is widely used to assess protein aggregation, fragmentation and conformational variants in the same solvents used during drug development and production. SV-AUC is especially useful for the analysis of drug substance, drug product and dosing solution, where other techniques may exhibit solvent matrix issues or concentration limitations. Recently, the only manufacturer of the analytical ultracentrifuge, released its newest (third generation) analytical ultracentrifuge, the Optima, in early 2017 to replace its aging 2nd generation XL series ultracentrifuges. However, SV-AUC data from four Optima units u...
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Biglycan is a small dermatan sulfate proteoglycan present in the extracellular matrix of a variety of connective tissues. Sedimentation velocity and equilibrium studies were carried out to determine the monomer molecular weight of... more
Biglycan is a small dermatan sulfate proteoglycan present in the extracellular matrix of a variety of connective tissues. Sedimentation velocity and equilibrium studies were carried out to determine the monomer molecular weight of biglycan in denaturing solvents and to define the oligomeric states of biglycan in physiologic solvents in the presence and absence of Zn2+. In 6 M guanidine chloride, biglycan is a monomer with s0(20,w) = 2.9 S and Mz = 93,100 (where Mz is z-average molecular weight). In 0.15 M NaCl, 50 mM Tris, pH 7.5, in the absence of divalent metal ions, and at concentrations above 1 mg/ml, biglycan is predominantly dimer (s0(20,w) = 4.8 S). Under these same conditions in solvent containing 5 mM Zn2+, biglycan exists predominantly as a hexamer, with s0(20,w) = 9.4 S and Mz approximately 600,000. In either case, the oligomers dissociate reversibly. In order to determine whether the glycosaminoglycan chains or the core protein was responsible for self-association, sedimentation velocity and sedimentation equilibrium studies were conducted on the isolated components. Dermatan sulfate chains prepared from biglycan, examined in both denaturing and physiologic solvents, show no significant difference in molecular weight (Mz approximately 22,000), whether or not the solvents contain Zn2+. However, biglycan core protein strongly self-associated in physiologic solvents. Thus, the self-association of biglycan appears to be mediated by the core protein and not by its glycosaminoglycan chains.
Research Interests: Chemistry, Biological Chemistry, Medicine, Biological Sciences, Animals, and 15 moreBiological, Zinc, CHEMICAL SCIENCES, Cattle, Proteoglycans, Glycosaminoglycans, Calorimetry, Molecular weight, Chondroitin Sulfate, Guanidines, Decorin, Edetic Acid, Extracellular Matrix Proteins, Dermatan sulfate, and Medical and Health Sciences
Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth... more
Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF tha...
Research Interests: Chemistry, Molecular Biology, Biology, Cell Adhesion, Medicine, and 15 moreHumans, Blood, Monoclonal Antibodies, Clinical Sciences, Neutrophils, Epidermal Growth Factor, Analytical Ultracentrifugation, Lectin, Molecular weight, Protein Binding, Ligands, Leukocytes, epitope, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products.... more
β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of β-lactoglobulin remains unclear. Sedimentation velocity experiments have identified new interactions between fluorescently-labelled β-lactoglobulin and other components in milk. Co-elution experiments support that these β-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. Ruminants (e.g. cows and goats) are born without circulating immunoglobulins, which they must obtain from their mothers' milk, whilst humans obtain immunoglobulins both through milk and during gestation via the placenta. We propose that β-lactoglobulin serves to pr...
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Publisher Summary This chapter discusses methods for detecting contamination of a protein sample by other proteins. Methods that provide molar quantitation of amino acids, specific prosthetic groups, or of active sites may be used to... more
Publisher Summary This chapter discusses methods for detecting contamination of a protein sample by other proteins. Methods that provide molar quantitation of amino acids, specific prosthetic groups, or of active sites may be used to assess the purity of a sample. In cases where the specific activity of the pure material is known, it may be appropriate to determine the unit activity as a measure of relative purity. The purity is then expressed as ratio of the amount measured to the amount expected. Good quantitation has been achieved using end-group analysis and quantitative analysis for specific prosthetic groups and for enzymatic activity. Electrophoretic methods provide some of the simplest, least expensive, and the most sensitive approaches to determine the number of protein components in a sample. Depending on the type of detection method available, nanogram to microgram quantities of sample are required. Because a contaminant constitutes a fixed weight fraction of a given sample, while the detection of a contaminant depends on the total mass of contaminant, the more sample applied to a gel, the better are the chances of detecting the contaminant.
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The diffusion interaction parameter (kD) has been demonstrated to be a high-throughput technique for characterizing interactions between proteins in solution. kD reflects both attractive and repulsive interactions, including long-ranged... more
The diffusion interaction parameter (kD) has been demonstrated to be a high-throughput technique for characterizing interactions between proteins in solution. kD reflects both attractive and repulsive interactions, including long-ranged electrostatic repulsions. Here, we plot the mutual diffusion coefficient (Dm) as a function of the experimentally determined Debye–Hückel–Henry surface charge (ZDHH) for seven human monoclonal antibodies (mAbs) in 15 mM histidine at pH 6. We find that graphs of Dm versus ZDHH intersect at ZDHH, ~ 2.6, independent of protein concentration. The same data plotted as kD versus ZDHH show a transition from net attractive to net repulsive interactions in the same region of the ZDHH intersection point. These data suggest that there is a minimum surface charge necessary on these mAbs needed to overcome attractive interactions.
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It has been known since the 1930’s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have... more
It has been known since the 1930’s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: 1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; 2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; 3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: 1) the intact IgG charges ranged from 0 to -13; 2) the F(ab’)2 fragments are nearly neutral for IgG1s and IgG2s, and about -5 for some of the IgG4s; 3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; 4...
Friedreich’s ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of... more
Friedreich’s ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K d, 4 mM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O 2. However, oxidation occurs rapidly and completely with H 2O 2 through a non-enzymatic process with a stoichiometry of two Fe(II)/H 2O 2, indicating complete reduction of H2O2 to H2O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemis...
Research Interests: Chemistry, Molecular Biology, Kinetics, Medicine, Molecular, and 15 moreIron Metabolism, Escherichia coli, Protein Aggregation, Iron Oxide, Iron, Oxidative Damage, Analytical Ultracentrifugation, Iron Toxicity, Isothermal Titration Calorimetry, Electron Paramagnetic Resonance, Frataxin, Biochemistry and cell biology, Iron binding proteins, Functional Properties, and binding sites
In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or closed-loop factors eIF4E, eIF4G, and PAB1. Using analytical ultracentrifugation with fluorescent detection system, we have identified a 57S... more
In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or closed-loop factors eIF4E, eIF4G, and PAB1. Using analytical ultracentrifugation with fluorescent detection system, we have identified a 57S translation complex that contains the 60S ribosome, mRNA, and the closed-loop factors. Previously published data by others also indicate the presence of a 50S-60S translation complex containing these same components. We have found that the abundance of this complex increased upon translational cessation, implying formation after ribosomal dissociation. Stoichiometric analyses of the abundances of the closed-loop components in the 57S complex indicate this complex is most similar to polysomal and monosomal translation complexes at the end of translation rather than at the beginning or middle of translation. In contrast, a 39S complex containing the 40S ribosome bound to mRNA and closed-loop factors was also identified with stoichiometries most simil...
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Every major biopharmaceutical company incorporates a protein crystallography unit that is central to its structure-based drug discovery efforts. Yet these capabilities are rarely leveraged toward the formal higher order structural... more
Every major biopharmaceutical company incorporates a protein crystallography unit that is central to its structure-based drug discovery efforts. Yet these capabilities are rarely leveraged toward the formal higher order structural characterization that is so challenging but integral to large-scale biologics manufacturing. Although the biotech industry laments the shortcomings of its favored biophysical techniques, x-ray crystallography is not even considered for drug development. Why not? We suggest that this is due, at least in part, to outdated thinking (for a recent industry-wide survey, see Gabrielson JP, Weiss IV WF. Technical decision-making with higher order structure data: starting a new dialogue. J Pharm Sci. 2015;104(4):1240-1245). We examine some myths surrounding protein crystallography and highlight the inherent properties of protein crystals (molecular identity, biochemical purity, conformational uniformity, and macromolecular crowding) as having practicable commonalit...
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As part of the Open AUC Project, the CFA is the next generation of analytical ultracentrifuge. The design philosophy of the CFA, which is to encourage hardware and software innovation, has been published. However, the hardware and... more
As part of the Open AUC Project, the CFA is the next generation of analytical ultracentrifuge. The design philosophy of the CFA, which is to encourage hardware and software innovation, has been published. However, the hardware and software that allow and encourage future development have not been described previously. Presented here is the CFA hardware and software architecture and the rationale for how this architecture was developed. Overall, both the hardware and software architecture is modular, allowing for updates and additions over time without the need for wholesale redevelopment. The common features needed by optical systems are contained in “stacks” of electronics for synchronizing the sources and detectors to the spinning rotor. Separate computer programs operate the stacks, the motion control, the centrifuge hardware, the experiment protocol, and each optical system. These programs communicate with one another to execute functions and to provide data and status information. Each program has a database associated with it to provide nonvolatile storage and inter-process communications. While the current implementation of the CFA uses one central computer to coordinate and operate all of the systems, the modular design includes provisions for using multiple computers should that be needed for a particular optical system.
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As interest in high-concentration protein formulations has increased, it has become apparent that routine, accurate protein charge measurements are necessary. There are several techniques for charge measurement, and a comparison of the... more
As interest in high-concentration protein formulations has increased, it has become apparent that routine, accurate protein charge measurements are necessary. There are several techniques for charge measurement, and a comparison of the methods is needed. The electrophoretic mobility, effective charge, and Debye-Hückel-Henry charge have been determined for bovine serum albumin, and human serum albumin. Three different electrophoretic methods were used to measure the electrophoretic mobility: capillary electrophoresis, electrophoretic light scattering, and membrane confined electrophoresis. In addition, the effective charge was measured directly using steady-state electrophoresis. Measurements made at different NaCl concentrations, pH, and temperatures allow comparison with previous charge estimates based on electrophoresis, Donnan equilibrium, and pH titration. Similar charge estimates are obtained by all of the methods. The strengths and limitations of each technique are discussed, as are some general considerations about protein charge and charge determination. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2123-2131, 2015.
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Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3 M) salt concentrations, in dilute (<0.1 M) salt solutions... more
Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3 M) salt concentrations, in dilute (<0.1 M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li(+), Na(+), K(+), Rb(+), Cs(+), GdnH(+), and Ca(2+)), identity. The Q* decreased in the order F(-) > Cl(-) > Br(-) > NO(3)(-) ∼ I(-) > SCN(-) > ClO(4)(-) ≫ SO(4)(2-), demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO(4)(2-) anion, despite being strongly h...
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Charge is a fundamental property of macromolecules. However, new instruments and new methods have been needed to explore the role of charge in determining the structure, stability, and interactions of macromolecules. An apparatus is... more
Charge is a fundamental property of macromolecules. However, new instruments and new methods have been needed to explore the role of charge in determining the structure, stability, and interactions of macromolecules. An apparatus is described here that is capable of performing equilibrium electrophoresis, electrophoretic mobility or diffusion measurements. This instrument acquires absorbance data from up to 512 positions along a quartz cell. The cell permits the establishment of an electric field along its length, while retaining macroions in the field of view. The prospects and limitations of using equilibrium electrophoresis for clinical applications are explored, particularly for characterizing macromolecular reagents. Applications are described for detecting charge heterogeneity, monitoring sample stability, and for determining the role of charge in molecular structure, stability and interactions. Because equilibrium electrophoresis provides little sample fractionation, the analysis of complex fluids requires the use of specific optical labels for discriminating components.
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Research Interests: Thermodynamics, Chemistry, Fluorescence, Biological Chemistry, Medicine, and 11 moreBiological Sciences, Humans, Biological, CHEMICAL SCIENCES, Protein Secondary Structure Prediction, Analytical Ultracentrifugation, Cysteine, Protein Quaternary Structure, Recombinant Proteins, Hydrogen-Ion Concentration, and Medical and Health Sciences
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Research Interests: Biochemistry, Chemistry, Physical Chemistry, Human Factors, Medicine, and 12 moreHumans, Viscosity, Temperature, Analytical Ultracentrifugation, Fluorescence polarization, Fluorescence anisotropy, Protein Conformation, Molecular weight, Protein Binding, Tissue Factor, Biochemistry and cell biology, and Medical biochemistry and metabolomics
We have investigated the formation of the aa-tRNA X EF-Tu X GTP ternary complex spectroscopically by monitoring a fluorescence change that accompanies the association of EF-Tu X GTP with Phe-tRNAPhe-F8, a functionally active analogue of... more
We have investigated the formation of the aa-tRNA X EF-Tu X GTP ternary complex spectroscopically by monitoring a fluorescence change that accompanies the association of EF-Tu X GTP with Phe-tRNAPhe-F8, a functionally active analogue of Phe-tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base. With this approach, the protein-nucleic acid interaction could be examined by direct means and at equilibrium. The fluorescence emission intensity of each Phe-tRNAPhe-F8 increased by 36-55% upon association with EF-Tu X GTP, depending on the solvent conditions. Thus, when Phe-tRNAPhe-F8 was titrated with EF-Tu X GTP, the extent of ternary complex formation was determined from the increase in emission intensity. A nonlinear least-squares analysis of the titration data yielded a dissociation constant of 0.85 nM for the ternary complex in 50 mM N-(2-hydroxyethyl)piperazine-N&#39;-2-ethanesulfonic acid (pH 7.6), 10 mM MgCl2, and 50 mM NH4Cl, at 6 degrees C. The delta H degrees of this interaction, determined by the temperature dependence of Kd, was -16 kcal/mol; the delta S degrees was therefore -16 cal mol-1 deg-1 at 6 degrees C in this buffer. In a more physiological polycation-containing solvent (&quot;polymix&quot;), the Kd was 4.7 nM. The ionic strength dependence of ternary complex formation showed that a minimum of two salt bridges and a substantial nonelectrostatic contribution are involved in the binding of aa-tRNA to EF-Tu. The affinities of unmodified aa-tRNAs for EF-Tu X GTP were determined by their abilities to compete with the fluorescent aa-tRNA for binding to the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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As a result of the renewed interest in analytical ultracentrifugation, the Beckman XL-A was released recently. This instrument automates spectrophotometric measurements of the concentration distribution of molecules in a gravitational... more
As a result of the renewed interest in analytical ultracentrifugation, the Beckman XL-A was released recently. This instrument automates spectrophotometric measurements of the concentration distribution of molecules in a gravitational field. Presented here is the design and the performance characteristics of a Rayleigh interferometer for the XL-A ultracentrifuge. The interferometer consists of a laser diode light source, imaging optics providing
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β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products.... more
β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of β-lactoglobulin remains unclear. Using the fluorescence-detection system within the analytical ultracentrifuge, we observed an interaction involving fluorescently labelled β-lactoglobulin in its native environment, i.e. cow and goat milk, for the first time. Co-elution experiments support that these β-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. The identification of these interactions, made possible through the use of fluorescence-detected analytical ultracentrifugation, provides possible clues to the long debated physiological function of this abundant milk protein.
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Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s... more
Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 t...
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Weak protein-protein interactions may be important to binding cooperativity. A panel of 7 fluorescently labelled tracer monoclonal IgG antibodies, differing in variable (V) and constant (C) region sequences, were sedimented in increasing... more
Weak protein-protein interactions may be important to binding cooperativity. A panel of 7 fluorescently labelled tracer monoclonal IgG antibodies, differing in variable (V) and constant (C) region sequences, were sedimented in increasing concentrations of unlabeled IgGs of identical, similar, and different backgrounds. Weak IgG::IgG attractive interactions were detected and characterized by global analysis of the hydrodynamic nonideality coefficient, k . The effects of salt concentration and temperature on k suggest the interactions are predominantly enthalpic in origin. The interactions were found to be variable in strength, affected by both the variable and constant regions, but indiscriminate with respect to IgG subclass. Furthermore, weak attractive interactions were observed for all the mAbs with freshly purified human poly-IgG. The universality of the weak interactions suggest that they may contribute to effector function cooperativity in the normal immune response, and we pos...
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The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used... more
The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used analytical ultracentrifugation with fluorescent detection system to identify the protein complexome of eRF1 in the yeast Saccharomyces cerevisiae. In addition to eRF1 presence in translating polysomes, we found that eRF1 associated with five other macromolecular complexes: 77S, 57S, 39S, 28S, and 20S in size. Generally equal abundances of each of these complexes were found. The 77S complex primarily contained the free 80S ribosome consistent with in vitro studies and did not appear to contain significant levels of the monosomal translating complex that co-migrates with the free 80S ribosome. The 57S and 39S complexes represented, respectively, free 60S and 40S ribosomal subunits bound to eRF1, associations not previously reported. The novel 28S and 20S ...
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▪ Analytical ultracentrifugation is a classical method of biochemistry and molecular biology. Because it is a primary technique, sedimentation can provide first-principle hydrodynamic and first-principle thermodynamic information for... more
▪ Analytical ultracentrifugation is a classical method of biochemistry and molecular biology. Because it is a primary technique, sedimentation can provide first-principle hydrodynamic and first-principle thermodynamic information for nearly any molecule, in a wide range of solvents and over a wide range of solute concentrations. For many questions, it is the technique of choice. This review stresses what information is available from analytical ultracentrifugation and how that information is being extracted and used in contemporary applications.
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A central dogma in immunology is that an antibody's in vivo functionality is mediated by two independent events: antigen binding by the variable (V) region, followed by effector activation by the constant (C) region. However, this... more
A central dogma in immunology is that an antibody's in vivo functionality is mediated by two independent events: antigen binding by the variable (V) region, followed by effector activation by the constant (C) region. However, this view has recently been challenged by reports suggesting allostery exists between the two regions, triggered by conformational changes or configurational differences. The possibility of allosteric signals propagating through the IgG domains complicates our understanding of the antibody structure-function relationship, and challenges the current subclass selection process in therapeutic antibody design. Here we review the types of cooperativity in IgG molecules by examining evidence for and against allosteric cooperativity in both Fab and Fc domains and the characteristics of associative cooperativity in effector system activation. We investigate the origin and the mechanism of allostery with an emphasis on the C-region-mediated effects on both V and C r...
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At least nine neurodegenerative diseases have been identified that are caused by the aggregation induced by long tracts of glutamine sequences. One such polyglutamine-containing protein is huntingtin, which is the primary factor... more
At least nine neurodegenerative diseases have been identified that are caused by the aggregation induced by long tracts of glutamine sequences. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington's disease. Sedimentation velocity with fluorescence detection is applied to carry out a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and SDD-AGE, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble incl...
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Phospholipids (PLs) are a major, diverse constituent of cell membranes. PL diversity arises from the nature of the fatty acid chains, as well as the headgroup structure. The headgroup charge is thought to contribute to both the strength... more
Phospholipids (PLs) are a major, diverse constituent of cell membranes. PL diversity arises from the nature of the fatty acid chains, as well as the headgroup structure. The headgroup charge is thought to contribute to both the strength and specificity of protein-membrane interactions. Because it has been difficult to measure membrane charge, ascertaining the role charge plays in these interactions has been challenging. Presented here are charge measurements on lipid Nanodiscs at 20°C in 100 mM NaCl, 50 mM Tris, at pH 7.4. Values are also reported for measurements made in the presence of Ca(2+) and Mg(2+) as a function of NaCl concentration, pH, and temperature, and in solvents containing other types of cations and anions. Measurements were made for neutral (phosphatidylcholine and phosphatidylethanolamine) and anionic (phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylinositol 4,5-bisphosphate (PIP2)) PLs containing palmitoyl-oleoyl and dimyristoyl fatty acid chai...
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Huntington's disease (HD) results from expansions of polyglutamine stretches (polyQ) in the huntingtin protein (Htt) that promote protein aggregation, neurodegeneration, and death. Since the diversity and sizes of the soluble... more
Huntington's disease (HD) results from expansions of polyglutamine stretches (polyQ) in the huntingtin protein (Htt) that promote protein aggregation, neurodegeneration, and death. Since the diversity and sizes of the soluble Htt-polyQ aggregates that have been linked to cytotoxicity are unknown, we investigated soluble Htt-polyQ aggregates using analytical ultracentrifugation. Soon after induction in a yeast HD model system, non-toxic Htt-25Q and cytotoxic Htt-103Q both formed soluble aggregates 29S to 200S in size. Because current models indicate that Htt-25Q does not form soluble aggregates, reevaluation of previous studies may be necessary. Only Htt-103Q aggregation behavior changed, however, with time. At 6 hr mid-sized aggregates (33S to 84S) and large aggregates (greater than 100S) became present while at 24 hr primarily only mid-sized aggregates (20S to 80S) existed. Multiple factors that decreased cytotoxicity of Htt-103Q (changing the length of or sequences adjacent to...
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Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an... more
Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting a...
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This work explores the heterogeneity of aggregation of polyglutamine fusion constructs in crude extracts of transgenic Caenorhabditis elegans animals. The work takes advantage of the recent technical advances in fluorescence detection for... more
This work explores the heterogeneity of aggregation of polyglutamine fusion constructs in crude extracts of transgenic Caenorhabditis elegans animals. The work takes advantage of the recent technical advances in fluorescence detection for the analytical ultracentrifuge. Further, new sedimentation velocity methods, such as the multi-speed method for data capture and wide distribution analysis for data analysis, are applied to improve the resolution of the measures of heterogeneity over a wide range of sizes. The focus here is to test the ability to measure sedimentation of polyglutamine aggregates in complex mixtures as a prelude to future studies that will explore the effects of genetic manipulation and environment on aggregation and toxicity. Using sedimentation velocity methods, we can detect a wide range of aggregates, ranging from robust analysis of the monomer species, through an intermediate and quite heterogeneous population of oligomeric species, and all the way up to detect...
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For over 75 years, analytical ultracentrifugation (AUC) has proved to be a powerful method for characterizing solutions of macromolecules and an indispensable tool for the quantitative analysis of macromolecular interactions [1–8]. It can... more
For over 75 years, analytical ultracentrifugation (AUC) has proved to be a powerful method for characterizing solutions of macromolecules and an indispensable tool for the quantitative analysis of macromolecular interactions [1–8]. It can be used to analyze the solution behavior of a variety of molecules in a wide range of solvents and over a wide range of solute concentrations. During analytical ultracentrifugation, samples are characterized in their native state under biologically relevant solution conditions, and in the absence of a matrix. Because AUC is nondestructive, samples may be recovered for further tests following the AUC procedure. Two complementary views of solution behavior are available from AUC. Sedimentation velocity provides first-principle, hydrodynamic information about the size and shape of molecules [1,4,6], while sedimentation equilibrium provides first-principle, thermodynamic information about the solution molar masses, stoichiometries, association constants, and solution nonideality [3,6,8]. Different experimental protocols are used to conduct these two types of analysis. This chapter covers the fundamentals of sedimentation velocity.
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Analytical ultracentrifugation (AUC) is a first principle method for characterizing the thermodynamics of macromolecules in solution. Since AUC directly assesses mass, it is particularly useful for characterizing both reversible and... more
Analytical ultracentrifugation (AUC) is a first principle method for characterizing the thermodynamics of macromolecules in solution. Since AUC directly assesses mass, it is particularly useful for characterizing both reversible and irreversible binding interactions between macromolecules. The principle measurement in AUC is the concentration as a function of radial position, which may be provided by either absorbance, interference or fluorescence detection. Each of these three different detectors may be used to characterize protein associations using either sedimentation equilibrium or sedimentation velocity analysis. Examples will be shown for characterizing irreversible (aggregate) formation, high-accuracy reversible association analysis, and the detection of protein interactions in complex and concentrated fluids (e.g. serum, cell cytosol).
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Analytical ultracentrifugation can provide useful thermodynamic and hydrodynamic information for a wide variety of chemical systems. It is apparent that through the use of modern electronics and computers, analytical ultracentrifugation... more
Analytical ultracentrifugation can provide useful thermodynamic and hydrodynamic information for a wide variety of chemical systems. It is apparent that through the use of modern electronics and computers, analytical ultracentrifugation is undergoing a renaissance. A modernization of ultracentrifugation is underway. The automation of acquisition, reduction and analysis of data has been quite successful. However, what has been more difficult to automate is the art of determining how much information is accessible from an experiment. This is a problem that becomes apparent to anyone starting a project that involves analytical sedimentation.