Delineation of the dynamic properties of individual lipid species in native and synthetic pulmonary surfactants.

Pulmonary surfactant (PS) is characterized by a highly conserved lipid composition and the formation of unique multilamellar structures within the lung. An unusually high concentration of DPPC is a hallmark of PS and is critical to the formation of a high surface area, stable air/water interface; the unusual lipid polymorphisms observed in PS are dependent on surfactant proteins, particularly lung surfactant protein B (SP-B). The molecular mechanisms of lipid trafficking and assembly in PS remain largely uncharacterized. Using (2)H and (31)P NMR, we characterize the dynamics and polymorphisms of the major lipid species in native PS and synthetic lipid mixtures as a function of SP-B1-25 addition. Our findings point to increased dynamics and a departure from a lamellar behavior for DPPC on addition of the peptide, consistent with our observations of DPPC phase separation in native surfactant. The monounsaturated lipids POPC, POPG and POPE remain in a lamellar phase and are less affected than DPPC by surfactant peptide addition. Additionally, we demonstrate that the properties of a native PS can be successfully mimicked by using a fully synthetic lipid mixture allowing the efficient evaluation of peptidomimetics under development for PS replacement therapies via NMR spectroscopy. The specificity of the dynamic changes in DPPC relative to POPC suggests the importance of tuning partitioning properties in successful peptidomimetic design.

Residue specific partitioning of KL4 into phospholipid bilayers.

KL4, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL4 and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL4 on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL4, and the partitioning depths of specific KL4 residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL4 induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL4 (V.C. Antharam et al., 2009), we are able to distinguish how KL4 affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL4 also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL4 are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.

Lipid polymorphism induced by surfactant peptide SP-B(1-25).

Pulmonary surfactant protein B (SP-B) is an essential protein for lowering surface tension in the alveoli. SP-B(1-25), a peptide comprised of the N-terminal 25 amino-acid residues of SP-B, is known to retain much of the biological activity of SP-B. Circular dichroism has shown that when SP-B(1-25) interacts with negatively charged lipid vesicles, it contains significant helical structure for the lipid compositions and peptide/lipid ratios studied here. The effect of SP-B(1-25) on lipid organization and polymorphisms was investigated via DSC, dynamic light scattering, transmission electron microscopy, and solid-state NMR spectroscopy. At 1-3 mol% peptide and physiologic temperature, SP-B(1-25) partitions at the interface of negatively charged PC/PG lipid bilayers. In lipid mixtures containing 1-5 mol% peptide, the structure of SP-B(1-25) remains constant, but (2)H and (31)P NMR spectra show the presence of an isotropic lipid phase in exchange with the lamellar phase below the T(m) of the lipids. This behavior is observed for both DPPC/POPG and POPC/POPG lipid mixtures as well as for both the PC and PG components of the mixtures. For 1-3 mol% SP-B(1-25), a return to a single lamellar phase above the lipid mixture T(m) is observed, but for 5 mol% SP-B(1-25) a significant isotropic component is observed at physiologic temperatures for DPPC and exchange broadening is observed in (2)H and (31)P NMR spectra of the other lipid components in the two mixtures. DLS and TEM rule out the formation of micellar structures and suggest that SP-B(1-25) promotes the formation of a fluid isotropic phase. The ability of SP-B(1-25) to fuse lipid lamellae via this mechanism, particularly those enriched in DPPC, suggests a specific role for the highly conserved N-terminus of SP-B in the packing of lipid lamellae into surfactant lamellar bodies or in stabilizing multilayer structures at the air-liquid interface. Importantly, this behavior has not been seen for the other SP-B fragments of SP-B(8-25) and SP-B(59-80), indicating a critical role for the proline rich first seven amino acids in this protein.

Partitioning, dynamics, and orientation of lung surfactant peptide KL(4) in phospholipid bilayers.

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL(4) is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL(4) 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 KL(4) in phospholipid bilayers, we introduced CD(3)-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via (2)H 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 side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL(4) in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL(4) 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 KL(4) relative to antimicrobial amphipathic alpha-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 KL(4). The unusual secondary structure of KL(4) and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.

Penetration depth of surfactant peptide KL4 into membranes is determined by fatty acid saturation.

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.

Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation.

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and (31)P and (2)H solid-state NMR spectroscopy. SP-B(59-80) forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B(59-80) in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B(59-80); in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B(59-80) penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL(4), a peptide mimetic of SP-B which was originally designed using SP-B(59-80) as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.

The helical structure of surfactant peptide KL4 when bound to POPC: POPG lipid vesicles.

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 alpha-helix; qualitative measurements using Raman, CD, 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 (phi, psi) angles that differ substantially from common values for alpha-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 alpha-helical structure allows KL 4 to form an amphipathic helix at the lipid interface.

Determination of peptide backbone torsion angles using double-quantum dipolar recoupling solid-state NMR spectroscopy.

Several approaches for utilizing dipolar recoupling solid-state NMR (ssNMR) techniques to determine local structure at high resolution in peptides and proteins have been developed. However, many of these techniques measure only one torsion angle or are accurate for only certain classes of secondary structure. Additionally, the efficiency with which these dipolar recoupling experiments suppress the deleterious effects of chemical shift anisotropy (CSA) at high magnetic field strengths varies. Dipolar recoupling with a windowless sequence (DRAWS) has proven to be an effective pulse sequence for exciting double-quantum (DQ) coherences between adjacent carbonyl carbons along the peptide backbone. By allowing this DQ coherence to evolve, it is possible to measure the relative orientations of the CSA tensors and subsequently use this information to determine the Ramachandran torsion angles phi and psi. Here, we explore the accuracies of the assumptions made in interpreting DQ-DRAWS data and demonstrate their fidelity in measuring torsion angles corresponding to a variety of secondary structures irrespective of hydrogen-bonding patterns. It is shown how a simple choice of isotopic labels and experimental conditions allows accurate measurement of backbone secondary structures without any prior knowledge. This approach is considerably more sensitive for determining structure in helices and has comparable accuracy for beta-sheet and extended conformations relative to other methods. We also illustrate the ability of DQ-DRAWS to distinguish between structures in heterogeneous samples.

A high resolution structure of the putative hinge region in M2 channel-lining segments of the nicotinic acetylcholine receptor.

Transmembrane peptide helices play key roles in signal transduction across cell membranes, yet little is known about their high-resolution structure or the role membrane composition plays in their association, structure, dynamics and ultimately their performance. Using magic angle spinning (MAS) homonuclear dipolar recoupling experiments, the backbone structure at positions L10, L11, and A12 of the M2 ion channel peptide was determined in two lipid systems. Their measurements are in agreement with M2 forming transmembrane helices, but the torsion angles vary considerably from common alpha-helical values. These measurements show remarkable agreement with a previous computational model of M2 peptides forming a pore domain in which their helices are kinked near the central leucine, L11 [R. Sankararamakrishnan, C. Adcock, M.S.P. Sansom, The pore domain of the nicotinic acetylcholine receptor: Molecular modeling, pore dimensions, and electrostatics, Biophys. J. 71 (1996) 1659-1671]. The generation of high resolution data for transmembrane helices is of critical importance in refining structures for membrane protein and developing models of helix packing interactions.