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George M Thurston, Jeffrey Mills, Lea V Michel, Keith P Van Nostrand, David Barnard, Angel Payan, David S Ross, ; Nuclear magnetic resonance and quasielastic and static light scattering studies of bovine gammaB-crystallin solutions. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5880.
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© ARVO (1962-2015); The Authors (2016-present)
To help characterize gamma crystallin attractive interactions by jointly studying its rotational diffusion, translational diffusion, and thermodynamic compressibility at intermediate protein concentrations.
15N-labeled bovine gammaB crystallin, without histidine tag, was produced in transformed E Coli by recombinant means, and isolated using size-exclusion and cation-exchange chromatography. Protein was concentrated for measurements in 10% D2O 100mM sodium phosphate buffer, pH 7.1, with 20mM dithiothreitol to inhibit oxidation. We obtained concentration and temperature dependence rotational self-diffusion through analysis of NMR transverse (T2) and longitudinal (T1) relaxation time measurements. Translational collective diffusion was measured with use of quasielastic light scattering, and solution Rayleigh ratios at 632.8 nanometer light wavelength were measured using static light scattering.
Characteristic times for rotational diffusion of gammaB crystallin slowed from 9 nanoseconds to 13 nanoseconds, as concentration was increased from 0.25 millimolar to 2.6 millimolar. This slowing-down is well above effects due to solution viscosity increases in this concentration range. Likewise, the collective translational diffusion coefficient Dc slowed from 9 x 10^(-11) m^2/s to 6 x 10^(-11) m^2/s over the same concentration range, consistent with previous literature. The dependence of the Rayleigh ratio on concentration is consistent with attractive interactions, as characterized previously.
The measured slowing of rotational diffusion with increasing concentration provides a potentially useful signal of interprotein attractions in gammaB crystallin solutions, as do previous, analogous characterizations of translational collective diffusion and light scattering intensity. To fully interpret the combined data, development of theory is needed that can incorporate both orientation-dependent direct as well as hydrodynamic protein-protein interactions.
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