Specific Lipid Studies in Complex Membranes by Solid‐State NMR Spectroscopy

Abstract

Specific interactions with phospholipids are often critical for the function of proteins or drugs, but studying these interactions at high resolution remains difficult, especially in complex membranes that mimic biological conditions. In principle, molecular interactions with phospholipids could be directly probed by solid‐state NMR (ssNMR). However, due to the challenge to detect specific lipids in mixed liposomes and limited spectral sensitivity, ssNMR studies of specific lipids in complex membranes are scarce. Here, by using purified biological 13C,15N‐labeled phospholipids, we show that we can selectively detect traces of specific lipids in complex membranes. In combination with 1H‐detected ssNMR, we show that our approach provides unprecedented high‐resolution insights into the mechanisms of drugs that target specific lipids. This broadly applicable approach opens new opportunities for the molecular characterization of specific lipid interactions with proteins or drugs in complex fluid membranes.

Cooperative Gating of a K+ Channel by Unmodified Biological Anionic Lipids Viewed by Solid-State NMR Spectroscopy

“…Three acyl chain isomers were previously identified by van Beekveld et al in PG lipids isolated from S. simulans, which were used in the present work: unmethylated (∼5%), anteiso (∼80%), and iso acyl chains (∼15%) . Of these, unmethylated lipids were found as C16:0 or C18:0, anteiso acyl chains were identified as C15:0 and C17:0, whereas iso acyl chains were observed to be C14:0, C15:0, C16:0, and C:17:0.…”

“…Chemical shift assignment of 13 C-PG lipids in the presence (green) and absence (purple) of NA-KirBac1.1. (a) Chemical structure of the PG lipids with three different isomers: unmethylated, iso (methylated at ω-1), and anteiso (methylated at ω-2), which were previously identified via gas chromatography . (b) Acyl chain chemical shift assignments of the labeled PG in the presence (green) and absence (purple) of NA-KirBac1.1.…”

“…U- 13 C-PG lipids were purified from Staphylococcus simulans as described previously . We reconstituted these lipids into liposomes with and without NA-KirBac1.1 with a 9:1 ratio of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/PG (10% Χ PG ).…”

“…PG was isolated, and quality was assessed as described previously . In brief, Staphylococcus simulans was grown for 16 h at 37 °C and 220 rpm in Luria broth (LB) followed by resuspension of the appropriate amount of cells in isotopically enriched media to reach OD 600 = 0.05.…”

“…In this report, we use uniformly 13 C enriched natural PG lipids, which are otherwise unaltered, isolated from bacteria to determine the binding affinity and cooperativity in lipid gating of the bacterial Kir channel KirBac1.1 . Like other Kir channels, KirBac1.1 is activated by anionic lipids including PG. , Bilayer cholesterol will also either activate or deactivate the channel, depending on the concentration. − Anionic lipids activate KirBac1.1 by triggering a series of concerted motions and structural rearrangement in the transmembrane region along an allosteric network linking PG binding to channel activation .…”