Peptoid-based macrodiscs of variable lipid composition for structural studies of membrane proteins by oriented-sample solid-state NMR.

Solid-state Nuclear Magnetic Resonance (NMR) in combination with magnetically aligned discoidal lipid mimics allows for studying the conformations of membrane proteins in planar, lipid-rich bilayer environments and at the physiological temperature. We have recently demonstrated the general applicability of macrodiscs composed of DMPC lipids and peptoid belts, which yield magnetic alignment and NMR spectroscopic resolution comparable or superior to detergent-containing bicelles. Here we report on a considerable improvement in the magnetic alignment and NMR resolution of peptoid-based macrodiscs consisting of a mixture of the zwitterionic and negatively charged lipids (DMPC/DMPG at the 85% to 15% molar ratio).

Aligned peptoid-based macrodiscs for structural studies of membrane proteins by oriented-sample NMR.

Development of a robust, uniform, and magnetically orientable lipid mimetic will undoubtedly advance solid-state NMR of macroscopically aligned membrane proteins. Here, we report on a novel lipid membrane mimetic based on peptoid belts. The peptoids, composed of 15 residues, were synthesized by alternating N-(2-phenethyl)glycine with N-(2-carboxyethyl)glycine residues at a 2:1 molar ratio.

Study of Förster Resonance Energy Transfer to Lipid Domain Markers Ascertains Partitioning of Semisynthetic Lipidated N-Ras in Lipid Raft Nanodomains.

Cellular membranes are heterogeneous planar lipid bilayers displaying lateral phase separation with the nanometer-scale liquid-ordered phase (also known as "lipid rafts") surrounded by the liquid-disordered phase. Many membrane-associated proteins were found to permanently integrate into the lipid rafts, which is critical for their biological function. Isoforms H and N of Ras GTPase possess a unique ability to switch their lipid domain preference depending on the type of bound guanine nucleotide (GDP or GTP).

Application of methyl-TROSY to a large paramagnetic membrane protein without perdeuteration: C-MMTS-labeled NADPH-cytochrome P450 oxidoreductase.

NMR spectroscopy of membrane proteins involved in electron transport is difficult due to the presence of both the lipids and paramagnetic centers. Here we report the solution NMR study of the NADPH-cytochrome P450 oxidoreductase (POR) in its reduced and oxidized states. We interrogate POR, first, in its truncated soluble form (70 kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240 kDa).

Conformational States of Cytochrome P450 Oxidoreductase Evaluated by Förster Resonance Energy Transfer Using Ultrafast Transient Absorption Spectroscopy.

NADPH-cytochrome P450 oxidoreductase (CYPOR) was shown to undergo large conformational rearrangements in its functional cycle. Using a new Förster resonance energy transfer (FRET) approach based on femtosecond transient absorption spectroscopy (TA), we determined the donor-acceptor distance distribution in the reduced and oxidized states of CYPOR. The unmatched time resolution of TA allowed the quantitative assessment of the donor-acceptor FRET, indicating that CYPOR assumes a closed conformation in both reduced and oxidized states in the absence of the redox partner.

The Ras G Domain Lacks the Intrinsic Propensity to Form Dimers.

Ras GTPase is a molecular switch controlling a number of cellular pathways including growth, proliferation, differentiation, and apoptosis. Recent reports indicated that Ras undergoes dimerization at the membrane surface through protein-protein interactions. If firmly established this property of Ras would require profound reassessment of a large amount of published data and modification of the Ras signaling paradigm.