The direct analysis of sedimentation equilibrium results obtained with polymerizing systems

Milthorpe, Bruce; Jeffrey, Peter D.; Nichol, L.W.

doi:10.1016/0301-4622(75)80007-x

Cited by 97 publications

(66 citation statements)

References 21 publications

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“…1B) from the different loading concentrations overlapped closely, indicating that both chemical and sedimentation equilibrium had been attained. Thus, the samples were homogeneous, and there were no significant concentrations of incompetent species such as irreversibly formed aggregates or truncated species (16,17).…”

Section: Resultsmentioning

confidence: 99%

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Thermodynamic and Hydrodynamic Properties of Human Tropoelastin

Toonkool¹,

Regan²,

Kuchel³

et al. 2001

Journal of Biological Chemistry

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Tropoelastin is the soluble precursor of elastin that bestows tissue elasticity in vertebrates. Tropoelastin is soluble at 20°C in phosphate-buffered saline, pH 7.4, but at 37°C equilibrium is established between soluble protein and insoluble coacervate. Sedimentation equilibrium studies performed before (20°C) and after (37°C) coacervation showed that the soluble component was strictly monomeric. Sedimentation velocity experiments revealed that at both temperatures soluble tropoelastin exists as two independently sedimenting monomeric species present in approximately equal concentrations. Species 1 had a frictional ratio at both temperatures of ϳ2.2, suggesting a very highly expanded or asymmetric protein. Species 2 displayed a frictional ratio at 20°C of 1.4 that increased to 1.7 at 37°C, indicating a compact and symmetrical conformation that expanded or became asymmetric at the higher temperature. The slow interconversion of the two monomeric species contrasts with the rapid and reversible process of coacervation suggesting both efficiently incorporate into the coacervate. A hydrated protein of equivalent molecular weight modeled as a sphere and a flexible chain was predicted to have a frictional ratio of 1.2 and 1.6, respectively. Tropoelastin appeared as a single species when studied by pulsed field-gradient spinecho NMR, but computer modeling showed that the method was insensitive to the presence of two species of equal concentration having similar diffusion coefficients. Scintillation proximity assays using radiolabeled tropoelastin and sedimentation analysis showed that the coacervation at 37°C was a highly cooperative monomer-n-mer self-association. A critical concentration of 3.4 g/liter was obtained when the coacervate was modeled as a helical polymer formed from the monomers via oligomeric intermediates.Elastin forms a highly insoluble cross-linked extracellular matrix that is predominantly responsible for the elasticity of vertebrate tissue. The precursor of elastin, tropoelastin, is devoid of cross-links. Following secretion from the cell surface, tropoelastin undergoes coacervation, which is a process of selfassociation characterized by an inverse temperature transition (1). Tropoelastin is soluble in aqueous solution at room temperature in vitro, but upon raising the temperature to 37°C the solution becomes turbid as tropoelastin molecules associate to form large aggregates (2, 3). This process of coacervation results from multiple intermolecular interactions of the hydrophobic domains (3, 4). The tropoelastin coacervate is a thick viscoelastic phase that is not miscible with the overlying solution (5). On cooling to 20°C, the aggregates dissociate reversibly, and the solution turns clear. Alternating between hydrophobic domains in the protein are short sequences of amino acid residues that form the cross-linking domains (6). Coacervation may concentrate and align tropoelastin molecules prior to elastin formation via lysyl oxidase-mediated cross-linkage of the lysine residues that leads to a grow...

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Section: Resultsmentioning

confidence: 99%

“…The ⍀ function was used to test for sample homogeneity and attainment of chemical equilibrium (16,17). 2 ⍀ is defined (16) as indicated in Equation 1,…”

Section: Methodsmentioning

confidence: 99%

Thermodynamic and Hydrodynamic Properties of Human Tropoelastin

Toonkool¹,

Regan²,

Kuchel³

et al. 2001

Journal of Biological Chemistry

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“…However, because the omega procedure [18,241, from which the psi analysis evolved, has had such little impact north of the equator, it is appropriate to illustrate the application of the present approach to simulated sedimentation-equilibrium distributions of reversible 1 : 1 complex formation between two dissimilar macromolecular reactants.…”

Section: Resultsmentioning

confidence: 99%

An Ultracentrifugal Approach to Quantitative Characterization of the Molecular Assembly of a Physiological Electron‐Transfer Complex. The Interaction of Electron‐Transferring Flavoprotein with Trimethylamine Dehydrogenase

Wilson

Scrutton

Cölfen

et al. 1997

European Journal of Biochemistry

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The interaction between two physiological redox partners, trimethylamine dehydrogenase and electron-transferring flavoprotein, has been characterized quantitatively by analytical ultracentrifugation at 4°C. Analysis of sedimentation-equilibrium distributions obtained at 15000 rpm for mixtures in 10 mM potassium phosphate, pH 7.5, by means of the psi function [Wills, P. R., Jacobsen, M. P. & Winzor, D. J. (1996) Biopolymers 38, 119-1301 has yielded an intrinsic dissociation constant of 3-7 pM for the interaction of electron-transferring flavoprotein with two equivalent and independent sites on the homodimeric enzyme. This investigation indicates the potential of sedimentation equilibrium for the quantitative characterization of interactions between dissimilar macromolecules.Keywords: electron-transfer flavoprotein ; trimethylamine dehydrogenase ; protein interaction ; analytical ultracentrifugation.The coexistence of protein . protein complexes in dissociation equilibrium with their constitutive reactants is a general feature of interprotein-electron-transfer reactions. Whereas the specific interactions involved in the formation of tightly bound protein . protein complexes may be elucidated by NMR and Xray -crystallographic procedures, those responsible for the weaker electron-transfer complexes are poorly understood because of the inability of current structure-determination procedures to accommodate the lability of the weaker protein . protein complexes. It is thought that the components of the electron; transfer complex may associate initially in unreactive configurations via non-specific interactions, and undergo a diffusional search for the specific configuration commensurate with electron transfer, termed the speculative reduction-in-dimensionality principle. The relatively weak interaction between two physiological redox partners, trimethylamine (Me,N) dehydrogenase and its electron-transferring flavoprotein (ETF), forms an archetypal example that is an excellent model for studying interprotein electron transfer.Me,N dehydrogenase is a complex iron-sulfur flavoprotein that converts Me,N to dimethylamine and formaldehyde via the reaction [I] (CH,),N + H 2 0 -(CH,),NH + HCHO + 2e-+ 2H'.I n vivo, the electrons derived from this conversion are passed to ETF, thereby generating the oxidised form of the enzyme [2]. Both components of this redox system have been cloned and From the high-resolution structure of Me,N dehydrogenase

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“…the combination of Ω o , the ordinate intercept of the dependence of Ω A (r) upon C(r), with C(r F ) yielded the monomer activity at the reference radial position, z A (r F ), and hence z A (r) for all C (r) as z A (r F )ψ A (r) [9]. By analogy with the above reasoning for the psi analysis, the application of the same approach to sedimentation equilibrium distributions reflecting protein dimerization in the presence of a small cosolute requires the rewriting of Eq.…”

Section: The Omega Approachmentioning

confidence: 99%

“…Prior to development of the characterization of selfassociation based on ψ A (r) [4], the direct evaluation of K 2 for a single-solute system was effected by means of the omega function [9] …”

Section: The Omega Approachmentioning

confidence: 99%