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Updated: Mar 5, 2026
Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR
Published on: February 12, 2019
Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel
Kendra K Frederick1, Vladimir K Michaelis2,3, Marc A Caporini4
1Whitehead Institute for Biomedical Research, Cambridge, MA 02142; kendra.frederick@utsouthwestern.edu.
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 in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13C- and 15N-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.
Insights
Researchers investigated the structure of yeast prion protein Sup35NM fibrils. Combining specific labeling with dynamic nuclear polarization NMR revealed insights into amyloid architecture, showing it lacks a parallel in-register structure.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- The yeast prion protein Sup35NM forms self-propagating amyloid fibrils.
- The precise arrangement of monomers within Sup35NM fibrils remains debated, with proposed models including beta-helical and parallel in-register structures.
- Determining intermolecular contacts is crucial but challenged by chemical shift degeneracy in standard NMR experiments.
Purpose of the Study:
- To elucidate the monomer organization within Sup35NM amyloid fibrils.
- To overcome limitations of traditional NMR methods for studying large protein systems.
- To develop a sensitive approach for determining the architecture of polymeric protein structures.
Main Methods:
- Utilized a combination of specific and segmental isotopic labeling strategies.
- Employed dynamic nuclear polarization (DNP) NMR to enhance experimental sensitivity.
- Investigated an amyloid form of Sup35NM.
Main Results:
- The study successfully limited chemical shift degeneracy and increased experimental sensitivity.
- The employed methods allowed for the examination of Sup35NM amyloid structure.
- Architectural information was obtained, indicating the absence of a parallel in-register structure in the examined Sup35NM amyloid.
Conclusions:
- The combination of specific labeling and DNP NMR is effective for determining the architecture of amyloid proteins like Sup35NM.
- This approach provides valuable insights into the structural organization of complex protein systems.
- The findings contribute to understanding the structural diversity of yeast prion protein amyloids.
