Allowance for boundary sharpening in the determination of diffusion coefficients by sedimentation velocity: a historical perspective

Winzor, Donald J.; Scott, David J.

doi:10.1007/s12551-017-0384-1

Cited by 4 publications

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“…Compared to ideal sedimentation in dilute conditions, colloidal hydrodynamic interactions at high concentration cause both retardation and self-sharpening of the sedimentation boundaries. 33,42,43 By contrast, the dominant effect of self-association is the enhancement of sedimentation, while boundaries remain significantly sharpened under non-ideal conditions. Through a detailed analysis of both the boundary shapes and migration throughout the entire sedimentation process, the new c NI (s 0 ) method allows, for the first time, to unravel the two competing phenomena: polydispersity from oligomerization and/or aggregation, and non-ideal interactions in a mean-field approximation expressed through coefficients k S for sedimentation and k D for diffusion.…”

Section: Resultsmentioning

confidence: 99%

Measuring Ultra-Weak Protein Self-Association by Non-ideal Sedimentation Velocity

et al. 2019

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Ultra-weak self-association can govern the macroscopic solution behavior of concentrated macromolecular solutions ranging from food products to pharmaceutical formulations and the cytosol. For example, it can promote dynamic assembly of multi-protein signaling complexes, lead to intracellular liquid–liquid phase transitions, and seed crystallization or pathological aggregates. Unfortunately, weak self-association is technically extremely difficult to study, as it requires very high protein concentrations where short intermolecular distances cause strongly correlated particle motion. Additionally, protein samples near their solubility limit in vitro frequently show some degree of polydispersity. Here we exploit the strong mass-dependent separation of assemblies in the centrifugal field to study ultra-weak binding, using a sedimentation velocity technique that allows us to determine particle size distributions while accounting for colloidal hydrodynamic interactions and thermodynamic non-ideality (Chaturvedi, S. K.; et al. Nat. Commun.2018, 9, 4415; DOI: 10.1038/s41467-018-06902-x). We show that this approach, applied to self-associating proteins, can reveal a time-average association state for rapidly reversible self-associations from which the free energy of binding can be derived. The method is label-free and allows studying mid-sized proteins at millimolar protein concentrations in a wide range of solution conditions. We examine the performance of this method with hen egg lysozyme as a model system, reproducing its well-known ionic-strength-dependent weak self-association. The application to chicken γS-crystallin reveals weak monomer–dimer self-association with KD = 24 mM, corresponding to a standard free energy change of approximately −9 kJ/mol, which is a large contribution to the delicate balance of forces ensuring eye lens transparency.

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Section: Resultsmentioning

confidence: 99%

Measuring Ultra-Weak Protein Self-Association by Non-ideal Sedimentation Velocity

et al. 2019

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“…Fortunately, an erroneous magnitude for k s does not affect the value of s o , the parameter most commonly being sought because of its relevance to the prediction of protein shape from hydrodynamic parameters (Garcia de la Torre et al 2000; Garcia de la Torre and Harding, 2013), although k s , if measured correctly, is itself useful in the delineation of molecular shape. In the event that the extent of boundary spreading is being used to evaluate the translational diffusion coefficient D the magnitude of k s also becomes important because of its use to make quantitative allowance for the effects of boundary sharpening arising from the linear negative s-c dependence exhibited by globular proteins (Fujita 1956(Fujita , 1959Baldwin 1957;Van Holde 1960;Scott et al 2015;Winzor and Scott 2018;Chaturvedi et al 2018). In that regard, the recommendation that use of the commonly used SEDFIT program for determining protein molar mass from sedimentation velocity distributions be confined to the analysis of experiments with low loading concentrations (Schuck 2005) reflected the omission of any allowance for this boundary sharpening effect at that stagea point now emphasized in the recent study dealing with the consequences of hydrodynamic nonideality (Chaturvedi et al 2018).…”

Section: Summary Of Current Approaches To Quantifying S-c Dependencementioning

confidence: 99%

Quantifying the concentration dependence of sedimentation coefficients for globular macromolecules: a continuing age-old problem

et al. 2021

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This retrospective investigation has established that the early theoretical attempts to directly incorporate the consequences of radial dilution into expressions for variation of the sedimentation coefficient as a function of the loading concentration in sedimentation velocity experiments require concentration distributions exhibiting far greater precision than that achieved by the optical systems of past and current analytical ultracentrifuges. In terms of current methods of sedimentation coefficient measurement, until such improvement is made, the simplest procedure for quantifying linear s-c dependence (or linear concentration dependence of 1/s) for dilute systems therefore entails consideration of the sedimentation coefficient obtained by standard c(s), g*(s) or G(s) analysis) as an average parameter ($$ \overline{s} $$ s ¯ ) that pertains to the corresponding mean plateau concentration (following radial dilution) ($$ \overline{c} $$ c ¯ ) over the range of sedimentation velocity distributions used for the determination of $$ \overline{s} $$ s ¯ . The relation of this with current descriptions of the concentration dependence of the sedimentation and translational diffusion coefficients is considered, together with a suggestion for the necessary improvement in the optical system.

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Analysis of Macromolecular Size Distributions in Concentrated Solutions

Chaturvedi,

Schuck

2024

Chemistry Methods

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The solution state of macromolecules in concentrated solutions impacts fields ranging from cell biology, to colloid chemistry and engineering of protein pharmaceuticals. Dependent on the interplay between repulsive and weakly attractive forces, proteins may exhibit oligomerization, aggregation, crystallization, liquid‐liquid phase separation, or the formation of multiprotein complexes. The particle size‐distribution is a key characteristic, but difficult to determine when interparticle distances are on the order of their size and macromolecular motion is coupled through hydrodynamic interactions. Here we extend sedimentation velocity analytical ultracentrifugation to measure macromolecular size distributions under these conditions: We apply results from statistical fluid mechanics for the concentration‐dependence of hindered settling and diffusion, embedded in a mean‐field approximation that can resolve coupled sedimentation and diffusion processes of different sized species given experimental sedimentation data. This is combined with a description of transient optical aberrations from lensing in the refractive index gradients associated with sedimentation boundaries (Wiener skewing). We demonstrate this approach in the application to protein solutions with macromolecular volume fractions up to ≈10 %, for example, resolving monomers and dimers of albumin at 140 mg/ml. This enables size‐distribution analysis of proteins at concentrations of therapeutic antibody formations and close to physiological concentration in serum and cells.

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Allowance for boundary sharpening in the determination of diffusion coefficients by sedimentation velocity: a historical perspective

Cited by 4 publications

References 42 publications

Measuring Ultra-Weak Protein Self-Association by Non-ideal Sedimentation Velocity

Measuring Ultra-Weak Protein Self-Association by Non-ideal Sedimentation Velocity

Quantifying the concentration dependence of sedimentation coefficients for globular macromolecules: a continuing age-old problem

Analysis of Macromolecular Size Distributions in Concentrated Solutions

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