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Insights into unfolded protein states and GPCR function by solution NMR

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We use extensively residual dipolar coupling, paramagnetic labeling, hydrogen bond scalar couplings and other NMR and non-NMR parameters to characterize unfolded protein states in a quantitative manner [1-6]. Thus we have generated a minimal structural ensemble of urea-denatured ubiquitin by constrained structure calculations. The ensemble reveals about 10 % native-like local structure in parts of the molecule on the background of long-range Gaussian chain behavior, thereby resolving Levinthal’s paradox. Recent orthogonal single molecule FRET data show excellent agreement to this ensemble. Furthermore, we have obtained atomic details of the pressure-assisted, cold-denatured state of ubiquitin by high-resolution NMR techniques. This state has structural propensities, which are very similar to ubiquitin’s alcohol-denatured (A-) state.

G protein-coupled receptors (GPCRs) are physiologically important transmembrane signaling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood due to missing dynamical information. We have recently shown [7] that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey 1-adrenergic receptor. I will discuss insights from NMR on ligand recognition, thermostabilization, signal transduction and G-protein binding as well as recently developed strategies for economic isotope labeling in insect cells [8].

References

[1] Huang, J.-R., Grzesiek, S., J. Am. Chem. Soc. 2010, 132, 694–705.

[2] Nisius, L., Grzesiek, S., Nat Chem 2012, 4, 711–717.

[3] Vajpai, N., Nisius, L., Wiktor, M., Grzesiek, S., Proc. Natl. Acad. Sci. USA 2013 , 110, E368 –76.

[4] Leung, H. T. A., Bignucolo, O., Aregger, R., Dames, S. A., Mazur, A., Bernèche, S., Grzesiek, S., J. Chem. Theory Comput. 2016, 12, 383–394.

[5] Bignucolo, O., Leung, H. T. A., Grzesiek, S., Bernèche, S., J. Am. Chem. Soc. 2015, 137, 4300–4303.

[6] Aznauryan, M., Delgado, L., Soranno, A., Nettels, D., Huang, J.-R., Labhardt, A. M., Grzesiek, S., Schuler, B., Proc. Natl. Acad. Sci. USA 2016 , 113, E5389 –E5398.

[7] Isogai, S., Deupi, X., Opitz, C., Heydenreich, F. M., Tsai, C.-J., Brueckner, F., Schertler, G. F. X., Veprintsev, D. B., Grzesiek, S., Nature 2016, 530, 237–241.

[8] Opitz, C., Isogai, S., Grzesiek, S., J. Biomol. NMR 2015 , 62, 373–385.

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