Podkorytov, I. S.; Belousov, M.; Bondarev, S.; Kaempf, K.; Zhouravleva, G.; et. all Detection of Flexible Portion of Protein Chain in Sup35NM Amyloid Fibrils by Means of Diffusion-Filtered NMR Experiment. The FEBS Journal 2017, 284 (Suppl. 1), 215.
DOI: 10.1111/febs.14174.
The Saccharomyces cerevisiae protein Sup35p is a part of the translation termination complex. A prion form of this protein has been intensely studied as a model for disease in higher organisms. In particular, the amyloidogenic portion of this protein, Sup35NM, attracted significant attention. One of the questions that remain unresolved is the status of M domain within Sup35NM: is it an integral part of the fairly rigid fibril architecture, or does it remain partially disordered? Historically, two types of experiments have been used to target flexible elements of protein fibrils: (i) solid-state HSQC experiments under MAS conditions and (ii) solution-state HSQC experiments on static samples. In both cases it is usually assumed that all protein material in the sample is sequestered in the fibrils. Such assumption is, generally speaking, incorrect. Protein fibrils always exist in a state of dynamic equilibrium with monomeric (oligomeric) species. Our diffusion experiments on a solid-state sample of Sup35NM demonstrated that ca. 20% of the signal originates from monomers, characterized by high translational mobility, whereas the remaining 80% belongs to the flexible fibril tails. Furthermore, we have prepared a dilute solution sample of Sup35NM, where ca. 85% of the signal was associated with monomers and the remaining 15% represented the flexible fibril tails. A comprehensive series of diffusion and relaxation measurements on this sample produced a self-consistent picture of dynamic equilibrium involving monomers and fibrils in slow exchange with each other. The addition of the diffusion filter to the standard HSQC sequence has allowed us to isolate the signals from the flexible parts of the fibril. Finally, the use of Sup35NM sample selectively labeled with 15N in valine positions made it possible to delineate the boundary between the flexible and the rigid portions of the fibrils.
Tags: Kaempf, Podkorytov, Skrynnikov