A biradical-tagged phospholipid as a polarizing agent for solid-state MAS Dynamic Nuclear Polarization NMR of membrane proteins
- University of Guelph, ON (Canada)
- North Carolina State University, Raleigh, NC (United States)
- Bruker Biospin, Billerica, MA (United States)
- North Carolina State University, Raleigh, NC (United States); Bruker Biospin, Billerica, MA (United States)
- University of Guelph, ON (Canada); Bruker Biospin, Billerica, MA (United States)
We report a novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix. DNP enhancements measured at 400 MHz/263 GHz and 600 MHz/395 GHz showed a strong field dependence but remained moderate at both fields, and comparable to those obtained for PR covalently modified with ToSMTSL. Additional continuous wave (CW) X-band electron paramagnetic resonance (EPR) experiments with ToSMTSL-PTE in solutions and in lipid bilayers revealed that an unfavorable conformational change of the linker connecting mononitroxides could be one of the reasons for moderate DNP enhancements. Further, differential scanning calorimetry (DSC) and CW EPR experiments indicated an inhomogeneous distribution and/or a possibility of a partial aggregation of ToSMTSL-PTE in DMPC:DMPA bilayers when the concentration of the polarizing agent was increased to 20 mol% to maximize the DNP enhancement. Thus, conformational changes and an inhomogeneous distribution of the lipid-based biradicals in lipid bilayers emerged as important factors to consider for further development of this matrix-free approach for DNP of membrane proteins.
- Research Organization:
- North Carolina State University, Raleigh, NC (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); Natural Sciences and Engineering Research Council of Canada (NSERC); National Science Foundation (NSF); North Carolina Biotechnology Centre (NCBC)
- Grant/Contract Number:
- FG02-02ER15354; GM130821; RGPIN-2014-04547; RGPIN-2018-04397; RR023614; CHE-0840501; 2009-IDG-1015
- OSTI ID:
- 1609382
- Alternate ID(s):
- OSTI ID: 1547486
- Journal Information:
- Solid State Nuclear Magnetic Resonance, Vol. 100, Issue C; ISSN 0926-2040
- Publisher:
- ElsevierCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
Solid-state NMR spectroscopy based atomistic view of a membrane protein unfolding pathway
|
journal | August 2019 |
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