Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

Abstract

KL(4) is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL(4) binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL(4) is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL(4) is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL(4), and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL(4). Additionally, a clear effect of KL(4) on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL(4) causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL(4) to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment.

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.…”

Role of the N-Terminal Seven Residues of Surfactant Protein B (SP-B)

“…This is because a peptide positioned close to the lipid head groups can increase the spacing between lipids without constraining the motions of the acyl chains, that, with the increase in headgroup spacing, have more orientational freedom. By contrast, peptides that insert deeply into the bilayer have little effect on acyl chain order [63], [64] because the peptide itself confers stearic constraints on acyl chain motions. That Super Mini-B (SP-B (1–25,63–78)) is so effective at disrupting the order deep in the bilayer interior is thus an indication that the N-terminal 7 residues likely do not penetrate deeply enough to constrain the amplitude of lipid acyl chain reorientation.…”

A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins

“…Fourth, the greatly enhanced bulk 4 peptide backbone (blue) partitioned in a membrane; the structure and partitioning were modeled from ssNMR distance restraints and EPR power saturation data, respectively. [22][23][24][25][26][27][28][29][30] For quantification of KL 4 DNP enhancement, leucine 12 (red) was glycerol signals in the TOTAPOL sample diminishes the ability to clearly observe underlying lipid resonances at those frequencies; additionally, for high fields and currently achievable MAS rates at 100 K with a 3.2 mm rotor, glycerol spinning sidebands can further obscure other sample resonances. Table 1 summarizes the enhancement values determined for SL-lipid and TOTAPOL systems investigated here.…”

“…The partitioning and structure of KL 4 is sensitive to both the degree of lipid acyl chain packing in proteoliposomes as well as pH. [22][23][24][25]31] The standard protocol for DNP sample preparation with water-soluble biradicals uses 60 % glycerol as a glassing agent in the solvent; however, it has been shown that small molecule cryoprotectants may alter the membrane environment. [7] Given that the SL-lipid system does not require or contain any glycerol, the inclusion of glycerol in the TOTAPOL system is likely changing the lipid acyl chain packing within the lipid bilayers and affecting the conformational equilibrium KL 4 adopts; alternatively differences in the pH or dielectric gradient at the lipid bilayer interface could be affecting the partitioning and ultimately the structure of the peptide.…”

“…Results at 14.1 T demonstrate that appreciable DNP enhancements are achieved throughout the lipid bilayer and that a DNP enhancement factor of 8.9 is achieved for KL 4 , a 21 residue amphipathic peptide which deeply partitions into the hydrophobic core region of lipid bilayers. 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. These are characteristics that emulate the properties of nitroxide biradicals, such as TOTAPOL, used in DNP experiments to date.…”