Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Abstract

Summary Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL4 is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL4 is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL4 in phospholipid bilayers, we introduced CD3-enriched leucines at four positions along the peptide to serve as probes of sidechain dynamics via 2H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine sidechains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL4 in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine sidechain dynamics, is consistent with monomeric KL4 lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL4 relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL4. The unusual secondary structure of KL4 and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.

Lucinactant attenuates pulmonary inflammatory response, preserves lung structure, and improves physiologic outcomes in a preterm lamb model of RDS

“…Biophysical studies of KL 4 demonstrating its ability to differentially partition into lipid lamellae and alter curvature strain suggest a mechanism for KL 4 peptide-mediated lipid organization and trafficking within the dynamic lung environment (35,36). By associating with lipid membranes, KL 4 may cause cytoskeletal changes that could alter cell signaling potentially through inducing leucine zipper-related mechanisms of inhibiting AP-1 or NF-kB regulatory signaling pathways.…”

A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins

“…The advent of low-temperature (100 K) DNP MAS ssNMR spectroscopy, with sensitivity gains of up to two orders of magnitude, shows promise for overcoming this sensitivity bottleneck. Figure 1 shows a schematic of the sample preparation strategy using SL-lipids and the structure and depth of membrane partitioning of KL 4 , which has been previously characterized by NMR and EPR investigations [22][23][24][25] This particular combination of SL-lipids was chosen because the chemical connectivity and physical location of the radicals in each lipid species provides for close spatial proximity of the radicals while also enabling a perpendicular orientation between the g-tensors of the nitroxides. [8][9][10][11][12][13][14][15][16] The strength of the dipolar coupling between the tethered nitroxides, the relative perpendicular orientation of their two g-tensors, and the protonation of nearby substituents have all been cited as important factors impacting signal enhancements in DNP experiments.…”

Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides

“…2 H NMR has long been used to determine the degree to which amino acid side chain dynamics are sensitive to structural environment, for example, the perturbation of the motion of leucine side chains in collagen fibrils (30), the dynamic impact of polylysine adsorption onto silica and hydroxyapatite surfaces (31), and the dynamics of hydrophobic side chains in membrane-associated proteins (32)(33)(34). Additionally, 13 C-2 H NMR correlation techniques have been used by Reif and coworkers to identify the dynamics of deuterated valine and leucine side chains in uniformly 13 C-labeled micro-crystalline proteins (35,36).…”