Ultracentrifuge Studies With Rayleigh Interference Optics. Iii. Computational Methods Applied to High‐speed Sedimentation Equilibrium Experiments*

Teller, David C.; Horbett, Thomas A.; Richards, E. G.; Schachman, H. K.

doi:10.1111/j.1749-6632.1969.tb14033.x

Cited by 138 publications

(84 citation statements)

References 27 publications

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“…Protein concentration ( C T ) was plotted against radius ( r ) from the arbitrary radius point ( r 0 ). The C T versus r plots were analyzed to determine K D according to the theoretical equation for monomer–dimer equilibrium: where C M ( r 0 ) is the monomer concentration at r 0 , M is the molecular weight of monomer, ϕ = ω 2 (1−|gnρ)/(2 RT ), and R , T , and ω are the gas constant, absolute temperature, and rotor speed, respectively (Teller et al 1969). The measurements at three different protein concentrations under the same buffer conditions were carried out using a rotor with three cells, and the results at different concentrations were analyzed individually with Equation 2, and the average of the three values was presented.…”

Section: Methodsmentioning

confidence: 99%

Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3

2001

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Although bovine ␤-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO 4 was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO 4 , the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (⌬H D ) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric ␤-lactoglobulin at pH 3.0 with NaClO 4 . The net heat effects after subtraction of the heat of salt dilution, corresponding to ⌬H D , were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the ⌬H D value at 20°C and the heat capacity change (⌬C p ) of dimer formation. In both NaCl and NaClO 4 , the obtained ⌬C p value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed ⌬C p values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of ␤-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

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Section: Methodsmentioning

confidence: 99%

Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3

2001

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“…where r 0 is arbitrary radius point, and C M (r 0 ) is the monomer concentration at r 0 , M is the molecular weight of monomer, ϭ 2 (1 Ϫ )/(2RT), and R, T, and are the gas constant, absolute temperature, and rotor speed, respectively (20). The measurements at three different protein concentrations under the same buffer conditions were carried out using a rotor with three cells, and the results at different concentrations were analyzed by global fitting using Equation 1.…”

Section: Methodsmentioning

confidence: 99%

Manipulating Monomer-Dimer Equilibrium of Bovine β-Lactoglobulin by Amino Acid Substitution

Sakurai

Goto

2002

Journal of Biological Chemistry

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Bovine ␤-lactoglobulin, a major protein in cow's milk composed of nine ␤-strands (␤A-␤I) and one ␣-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0. It is assumed that the intermolecular ␤-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation. Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation. Various I-strand mutants showed decreases in K a , suggesting that the intermolecular ␤-sheet is essential for dimer formation. By substituting either Asp 33 or Arg 40 on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in K a was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg 40 on one monomer and Asp 33 on the other monomer in the wild-type dimer. However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg 33 and Asp 40 of different molecules. These results suggested that protein-protein interactions of bovine ␤-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.

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“…6). Although for absorption optics this involved an extrapolation and an evaluation of the baseline or background absorbance of non-sedimenting species – and does not create too much difficulty, for Rayleigh interference – where the optical records are of the solute concentration relative to a reference position, conventionally taken as the air/solution meniscus 10 – this involved either a rather complex mathematical manipulation of the data followed by an ill-conditioned extrapolation of two functions, based on a method of Teller et al 11 – the so-called intercept over slope method 7 or a separate experiment involving synthetic boundary cells 12 . We now present a completely new version of the program which (i) interfaces into the widely used SEDFIT platform for sedimentation analysis of macromolecules (ii) provides a much more rigorous method of obtaining the baseline and meniscus concentration for the Rayleigh interference optical system and (iii) provides an estimate for the distribution of molecular weight.…”

Section: Introductionmentioning

confidence: 99%

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

Schuck

Gillis

Besong

et al. 2014

Analyst

121

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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.

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Ultracentrifuge Studies With Rayleigh Interference Optics. Iii. Computational Methods Applied to High‐speed Sedimentation Equilibrium Experiments*

Cited by 138 publications

References 27 publications

Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3

Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3

Manipulating Monomer-Dimer Equilibrium of Bovine β-Lactoglobulin by Amino Acid Substitution

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

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