The Helical Structure of Surfactant Peptide KL₄ When Bound to POPC: POPG Lipid Vesicles

Abstract

KL 4 is a 21-residue peptide employed as a functional mimic of lung surfactant protein B which successfully lowers surface tension in the alveoli. A mechanistic understanding of how KL 4 affects lipid properties has proven elusive as the secondary structure of KL 4 in lipid preparations has not been determined at high resolution. The sequence of KL 4 is based on the C-terminus of SP-B, a naturally occurring helical protein that binds to lipid interfaces. The spacing of the lysine residues in KL 4 precludes the formation of a canonical amphipathic α-helix; qualitative measurements using Raman, CDc and FTIR spectroscopies have given conflicting results as to the secondary structure of the peptide as well as its orientation in the lipid environment. Here, we present a structural model of KL 4 bound to lipid bilayers based on solid state NMR data. Double-quantum correlation experiments employing 13 C-enriched peptides were used to quantitatively determine the backbone torsion angles in KL 4 at several positions. These measurements, coupled with CD experiments, verify the helical nature of KL 4 when bound to lipids, with (Φ, Ψ) angles that differ substantially from common values To whom correspondence should be addressed. Tel: 352-846-1506. Fax:352-392-3422. E-mail: jrlong@mbi.ufl for α-helices of (-60, -45). The average torsion angles found for KL 4 bound to POPC: POPG lipid vesicles are (-105, -30); this deviation from ideal α-helical structure allows KL 4 to form an amphipathic helix at the lipid interface.Keywords pulmonary surfactant; Surfactant Protein B; KL 4 ; sinapultide; lucinactant; peptide secondary structure; alpha-helix; hydrophobic moment; lipid bilayers; membrane proteins; phosphatidylcholine; phosphatidylglycerol; solid state NMR; dipolar recoupling; magic angle spinning Pulmonary surfactant is a lipid-rich fluid, containing key proteins, forming the inner mucosal lining of the alveoli. The primary functions of lung surfactant are to minimize surface tension at the alveolar air-fluid interface and provide a barrier against disease (1-5). Inadequate protein levels are a leading cause of respiratory distress syndrome (RDS) in premature infants (6-8).Current therapies for RDS primarily rely on administration of lung surfactant from exogenous sources (9-12). This reliance on xenogenic surfactant is due to the critical role of lung surfactant protein B (SP-B), a highly hydrophobic, 79 residue protein which functions as a homodimer containing 7 disulfide bridges (13). A multitude of roles for SP-B have been proposed and experimentally established. These include surface tension minimization, facilitation of lipid adsorption and resorption at the air-fluid interface, intracellular surfactant trafficking and improved respiratory dynamics in general (14).Synthetic, peptide-based lung surfactant replacements have received noticeable attention (15-17) as the use of synthetic analogs would remove the immunologic risks associated with animal-derived surfactant and allow for greater therapeutic consistency ...

An Open Label, Pilot Study of Aerosurf®Combined with nCPAP to Prevent RDS in Preterm Neonates

“…Lucinactant is a novel synthetic surfactant that contains KL 4 , a 21-amino acid peptide mimicking the function of surfactant protein B (SP-B). (13) The safety and effectiveness of lucinactant has been characterized in Phase 3 clinical trials for the prevention of RDS when administered as an intratracheal suspension. (14,15) Lucinactant may be uniquely suited for aerosolized delivery, as the KL 4 peptide contained in lucinactant is not susceptible to damage and inactivation during aerosolization.…”

Membrane Protein Structure Determination: Back to the Membrane

“…Resonances in these spectra can be assigned using NCACX and NCOCA experiments [69–74], complemented by 13 C- 13 C [75, 76] and 13 C- 15 N correlation experiments [77]. Two-dimensional 13 C- 13 C correlation MAS spectra of Ail and OmpX in proteoliposomes (Fig.…”

A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins

“…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.…”

“…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.…”

“…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.…”