Solid-State NMR Study of Amyloid Nanocrystals and Fibrils Formed by the Peptide GNNQQNY from Yeast Prion Protein Sup35p

Abstract

Sup35p is a prion protein found in yeast that contains a prion-forming domain characterized by a repetitive sequence rich in Gln, Asn, Tyr, and Gly amino acid residues. The peptide GNNQQNY7-13 is one of the shortest segments of this domain found to form amyloid fibrils, in a fashion similar to the protein itself. Upon dissolution in water, GNNQQNY displays a concentration-dependent polymorphism, forming monoclinic and orthorhombic crystals at low concentrations and amyloid fibrils at higher concentrations. We prepared nanocrystals of both space groups as well as fibril samples that reproducibly contain three (coexisting) structural forms and examined the specimens with magic angle spinning (MAS) solid-state nuclear magnetic resonance. 13C and 15N MAS spectra of both nanocrystals and fibrils reveal narrow resonances indicative of a high level of microscopic sample homogeneity that permitted resonance assignments of all five species. We observed variations in chemical shift among the three dominant forms of the fibrils which were indicated by the presence of three distinct, self-consistent sets of correlated NMR signals. Similarly, the monoclinic and orthorhombic crystals exhibit chemical shifts that differ from one another and from the fibrils. Collectively, the chemical shift data suggest that the peptide assumes five conformations in the crystals and fibrils that differ from one another in subtle but distinct ways. This includes variations in the mobility of the aromatic Tyr ring. The data also suggest that various structures assumed by the peptide may be correlated to the "steric zipper" observed in the monoclinic crystals.

250GHz CW gyrotron oscillator for dynamic nuclear polarization in biological solid state NMR

“…Two research areas in which high resolution MAS experiments have proved especially successful are studies of amyloid fibrils [37,[51][52][53][54][55][56][57] and membrane proteins [42,[58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75]. However, in both of these cases, low sensitivity currently limits the information that can be gleaned from the spectra.…”

Phosphorylation as Conformational Switch from the Native to Amyloid State: Trp-Cage as a Protein Aggregation Model

“…However, this concentration is similar to that reported to be essential for amyloid formation of the GNNQQNY core segment of yeast prion Sup35. 61 Using different mutants and phosphorylated serine variants, we gained site-specific information on the structure of the different forms of Trpcage and we could outline the aggregation mechanism. The CD and NMR results revealed that the phosphorylation of any of the serine residues destabilizes the native structure leading to a highly dynamic disordered chain (Table 1 and Fig.…”

Heteronuclear proton assisted recoupling

“…Similarly, one can deriveH (2) 2×2 -the second order contribution of term 2 in Eq. (5) with itself (i.e.…”

“…Autocross terms of the formH (2) j×j yield longitudinal T 10 operators that can be rearranged as combinations of ZQ/DQ fictitious spin operators. More precisely the relevant Hamiltonian can be written as three commuting terms…”

“…Recent applications of MAS NMR to such systems have provided insight into protein folding and misfolding, 1 amyloid aggregation, 2,3 and membrane protein function. 4 MAS NMR experiments are preferred for structural studies of these types of systems since they average second rank tensor interactions such as the chemical shift anisotropy and dipolar interactions 5 and therefore yield high resolution spectra.…”