REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins
 

REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

Publication date: April 2015
Source:Journal of Magnetic Resonance, Volume 253</br>
Author(s): Lihui Jia , Shuang Liang , Kelly Sackett , Li Xie , Ujjayini Ghosh , David P. Weliky</br>
Rotational-echo double-resonance (REDOR) solid-state NMR is applied to probe the membrane locations of specific residues of membrane proteins. Couplings are measured between protein 13CO nuclei and membrane lipid or cholesterol 2H and 31P nuclei. Specific 13CO labeling is used to enable unambiguous assignment and 2H labeling covers a small region of the lipid or cholesterol molecule. The 13CO–31P and 13CO–2H REDOR respectively probe proximity to the membrane headgroup region and proximity to specific insertion depths within the membrane hydrocarbon core. One strength of the REDOR approach is use of chemically-native proteins and membrane components. The conventional REDOR pulse sequence with 100kHz 2H ? pulses is robust with respect to the 2H quadrupolar anisotropy. The 2H T 1’s are comparable to the longer dephasing times (?’s) and this leads to exponential rather than sigmoidal REDOR buildups. The 13CO–2H buildups are well-fitted to A ×(1- e - ?? ) where A and ? are fitting parameters that are correlated as the fraction of molecules (A) with effective 13CO–2H coupling d =3?/2. The REDOR approach is applied to probe the membrane locations of the “fusion peptide” regions of the HIV gp41 and influenza virus hemagglutinin proteins which both catalyze joining of the viral and host cell membranes during initial infection of the cell. The HIV fusion peptide forms an intermolecular antiparallel ? sheet and the REDOR data support major deeply-inserted and minor shallowly-inserted molecular populations. A significant fraction of the influenza fusion peptide molecules form a tight hairpin with antiparallel N- and C-? helices and the REDOR data support a single peptide population with a deeply-inserted N-helix. The shared feature of deep insertion of the ? and ? fusion peptide structures may be relevant for fusion catalysis via the resultant local perturbation of the membrane bilayer. Future applications of the REDOR approach may include samples that contain cell membrane extracts and use of lower temperatures and dynamic nuclear polarization to reduce data acquisition times.
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