Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Abstract

The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a β-helical arrangement, whereas others suggest a parallel inregister organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 C-and 15 N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. (2), is an amyloid form of the translation termination factor Sup35 (3). The amyloid fold is polymeric fiber of protein monomers that is rich in β-sheets that run perpendicular to the fiber axis. Amyloid formation sequesters Sup35 from the ribosome, changing the rate of stop-codon read-through and creating a variety of new yeast phenotypes (4,5). Different regions of the Sup35 protein are responsible for prion templating, prion inheritance, and translation termination. The N-terminal domain "N" is Q/N-rich and required for the formation and templating of amyloid. The highly charged K/E-rich middle domain "M" promotes solubility in the nonprion form and is required for chaperone-mediated prion inheritance (6, 7). The C-terminal domain is necessary and sufficient for translation termination. The N and M domains together, NM, contain all of the necessary features to act as an amyloid-based prion.A wide variety of approaches have been used to investigate Sup35 prion structures, but a consensus structural picture has yet to emerge. Amyloids are notoriously difficult to study, mainly because they are insoluble yet noncrystalline and are therefore not easily amenable to traditional structural biology methods. In all models, at least some part of the N domain is involved in the amyloid fold (8-11), and most of the M domain is thought to be solvent-accessible and intrinsically disordered (11-13). However, there are two conflicting models of how monomers of NM are arranged in amyloid fibers. In one model, called the β-helical model, monomers make intermolecular contacts in discrete regions, with the region in-between making intramolecular contacts. This model is supported by the patterns of interaction and cross-linking determined from a series of site-directed mutants (8). The work has the caveat that mutations may perturb the fibril structure. However, fibers made from the mutated versions of the protein ca...

Proteinase K resistant cores of prions and amyloids

“…Then, it was shown by NMR that tyrosine and phenylalanine residues in the N domain, as well as some leucines in the M domain are located in-register at a distance of about 0.5 nm [57,61,62]. However, Sup35 amyloid that does not have a parallel in-register structure was also reported [63]. A three-rung β-solenoid model was also proposed, based on amino acid proximity studies using fluorescent labels [59].…”

Insights into protein misfolding and aggregation enabled by solid-state NMR spectroscopy

“…These peptide X-ray structures have yielded a systematic classification of amyloid-like β-sheet spine assemblies. Although it is dangerous to extrapolate the structures of the short-peptide crystals to the actual amyloid architecture of their parent proteins [78, 79], these X-ray data are valuable for ssNMR studies as high-resolution reference structures. The peptide crystals are also ideal to experimentally validate (novel) ssNMR measurements of amyloid structure.…”

NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein

“…Segmentally introducing isotopic labels into proteins also allows their NMR spectroscopic investigation by restricting the emerge of signals to a particular protein domain or region in the full-length protein context (Busche et al, 2009;Buchinger et al, 2010;Minato et al, 2012Minato et al, , 2017Shiraishi et al, 2018;Wiegand et al, 2018). Several methods, including expressed protein ligation (EPL), protein-trans splicing (PTS), fragment ligation using sortases or asparagine endopeptidases have been used for segmental isotopic labeling (Skrisovska and Allain, 2008;Muona et al, 2010;Freiburger et al, 2015;Kwon et al, 2015;Williams et al, 2016;Frederick et al, 2017;Mikula et al, 2017Mikula et al, , 2018Wiegand et al, 2018). Albeit these various available methods, segmental isotopic labeling for structural studies remains challenging due to the requirement of relatively high quantities (>mg scale) and high purity.…”

“…However, IDRs typically have low complexity sequences, and their NMR signals tend to degenerate due to the lack of a well-defined fold. The higher degeneracy of IDRs compared to highly structured proteins could hinder their detailed NMR analysis, calling for strategies to alleviate signal overlaps when studying IDPs and IDRs (Nabeshima et al, 2014;Frederick et al, 2017). We chose TonB from Gram-negative bacteria as an example of multidomain proteins where a central proline-rich low complexity region (LCCR) connects a globular C-terminal domain (CTD) to an N-terminal transmembrane (TM) domain (Figures 1, 2A, Figure S1).…”