TY - JOUR
T1 - Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register
AU - Frederick, Kendra K.
AU - Michaelis, Vladimir K.
AU - Caporini, Marc A.
AU - Andreas, Loren B.
AU - Debelouchina, Galia T.
AU - Griffin, Robert G.
AU - Lindquist, Susan
N1 - Funding Information:
We thank members of the S.L. and R.G.G. laboratories for valuable discussions during the course of this research. S.L. was a Howard Hughes Medical Institute Investigator. K.K.F. was an HHMI Fellow of the Life Science Research Foundation, and V.K.M. was a Banting Fellow of the Natural Science and Engineering Research Council of Canada, Government of Canada. This work was supported by NIH Grants GM-025874 (to S.L.) and EB-003151, EB-002804, and EB-002026 (to R.G.G.).
PY - 2017/4/4
Y1 - 2017/4/4
N2 - 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 â-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 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.
AB - 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 â-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 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.
KW - Amyloid
KW - Dynamic nuclear polarization
KW - Solid-state NMR
KW - Sup35
KW - [PSI] prion
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U2 - 10.1073/pnas.1619051114
DO - 10.1073/pnas.1619051114
M3 - Article
C2 - 28330994
AN - SCOPUS:85016969397
SN - 0027-8424
VL - 114
SP - 3642
EP - 3647
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 14
ER -