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George M Thurston, Jeffrey L. Mills, Kaylee L. Mathews, John Zanet, Angel Payan, Lea V. Michel, Keith Van Nostrand, Christopher W. Wahle, John F. Hamilton, David S. Ross; Concentration- and temperature-dependent chemical shift perturbations in solutions of bovine GammaB-crystallin. Invest. Ophthalmol. Vis. Sci. 2016;57(12):731.
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© ARVO (1962-2015); The Authors (2016-present)
To use concentration- and temperature-dependent chemical shift perturbations (CSPs) as an aid for studying anisotropic, orientation-dependent interactions between GammaB-crystallin proteins in solution, with the longer-term aim of quantifying how key combinations of interacting residues affect liquid-liquid phase separation temperature and light scattering.
We produced 13C- and/or 15N-labeled recombinant GammaB for the joint purposes of assigning chemical shifts, obtaining an NMR solution structure, and studying the CSPs obtained from two-dimensional heteronuclear spin quantum coherence (HSQC) experiments. We examined spectra on backbone nitrogen and attached hydrogen nuclei for CSPs, up to 3% volume fraction and at temperatures of 25C and 15C, using a 900MHz NMR instrument at the NMRFAM facility in Madison, Wisconsin.
Increasing GammaB-crystallin concentration to 3% by volume produces a progressive spread of the cumulative distribution function of the magnitude of 15N backbone CSPs, over a range of about 0.012 parts per million. The HSQC peaks that were most highly perturbed with increasing protein concentration are typically quite close to charged residues, and many are close to locations at which intermolecular contacts occur in GammaB crystals. Concentration-dependent CSPs at 15C and 25C were positively correlated. The range of the measured 15N backbone CSPs that occurred upon comparing the 15C and 25C HSQC spectra was about 5 times larger than the corresponding range of shifts with concentration. The CSPs that occurred upon reducing temperature were negatively correlated with those that occurred upon increasing concentration.
The backbone residues having the most prominent chemical shift perturbations underline the role of electrostatic interactions for understanding GammaB-GammaB interactions. Because a predominant source of CSPs is the local electric field, which is altered by protein proximity, the locations and magnitudes of concentration-dependent CSPs can provide useful input to calibrate and test models of electrostatic and other protein interactions. With further modeling the CSPs may thereby help quantitatively rank the relative importance of selected relative protein orientations, and by extension individual residues, in producing the observed phase behavior and light scattering.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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