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

Abstract

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 100 kHz 2H π pulses is robust with respect to the 2H quadrupolar anisotropy. The 2H T1’s are comparable to the longer dephasing times (τ’s) and this leads to exponential rather than sigmoidal REDOR buildups. The 13CO-2H buildups are well-fitted to A × (1 − e−γτ) where A and γ are fitting parameters that are correlated as the fraction of molecules (A) with effective 13CO-2H coupling d = 3γ/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 β 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- α helices and the REDOR data support a single peptide population with a deeply-inserted N-helix. The shared feature of deep insertion of the β and α 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.

Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes

“…To conduct this test, we used rotational echo double-resonance (REDOR) NMR spectroscopy, which measures the dipolar coupling between 13 C and 2 H nuclei. 13 C{ 2 H} REDOR is commonly used to probe protein-membrane interactions (31)(32)(33)(34). We found that leucine interacts with a hydrogen near the headgroup of decanoic acid but not at the end of the tail ( Fig.…”

Efficient Fusion at Neutral pH by Human Immunodeficiency Virus gp41 Trimers Containing the Fusion Peptide and Transmembrane Domains

“…45 FP insertion in a single leaflet is evidenced by NMR contacts between multiple FP residues and lipid tails. 59 There is not close contact between the FP and the MPER or between the FP and the TM, as evidenced by absence of NMR crosspeaks between these domains. 34…”

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

“…In summary, while the I=1/2 spin system suffers from lower sensitivity for this three-pulse ESEEM technique when compared to the I=1 for 2 H, 13 C has been able to be successfully used as the NMR-active nuclei in order to determine local secondary structure of a model membrane peptide for the first time. This technique using 13 C-labeled amino acids is comparable to REDOR solid-state NMR spectroscopy techniques; however, it requires less sample concentration (μM) and shorter data acquisition times (~30 minutes) making it ideal for studying membrane protein secondary structure [34,35]. This method provides an alternative approach to this biophysical technique in order to provide more versatility and options when used to study a variety of secondary structures.…”