An AFM study of solid-phase bilayers of unsaturated PC lipids and the lateral distribution of the transmembrane model peptide WALP23 in these bilayers

An altered lipid packing can have a large influence on the properties of the membrane and the lateral distribution of proteins and/or peptides that are associated with the bilayer. Here, it is shown by contact-mode atomic force microscopy that the surface topography of solid-phase bilayers of PC lipids with an unsaturated cis bond in their acyl chains shows surfaces with a large number of line-type packing defects, in contrast to the much smoother surfaces observed for saturated PC lipids. Di-n:1-PC (n = 20, 22, 24) and (16:0,18:1)-PC (POPC) were used. Next, the influence of an altered lipid environment on the lateral distribution of the single α-helical model peptide WALP23 was studied by incorporating the peptide in the bilayers of di-n:1-PC (n = 20, 22, 24) and (16:0,18:1)-PC unsaturated lipids. The presence of WALP23 leads to an increase in the number of packing defects but does not lead to the formation of the striated domains that were previously observed in bilayers of saturated PC lipids and WALP. This is ascribed to the less efficient lateral lipid packing of the unsaturated lipids, while the increase in packing defects is probably an indirect effect of the peptide. Finally, the fact that an altered lipid packing affects the distribution of WALP23 is also confirmed in an additional experiment where the solvent TFE (2,2,2-trifluorethanol) is added to bilayers of di-16:0-PC/WALP23. At 3.5 vol% TFE, the previous striated ordering of the peptide is abolished and replaced by loose lines

Orientation and dynamics of transmembrane peptides: the power of simple models

In this review we discuss recent insights obtained from well-characterized model systems into the factors that determine the orientation and tilt angles of transmembrane peptides in lipid bilayers. We will compare tilt angles of synthetic peptides with those of natural peptides and proteins, and we will discuss how tilt can be modulated by hydrophobic mismatch between the thickness of the bilayer and the length of the membrane spanning part of the peptide or protein. In particular, we will focus on results obtained on tryptophan-flanked model peptides (WALP peptides) as a case study to illustrate possible consequences of hydrophobic mismatch in molecular detail and to highlight the importance of peptide dynamics for the experimental determination of tilt angles. We will conclude with discussing some future prospects and challenges concerning the use of simple peptide/lipid model systems as a tool to understand membrane structure and function

Evidence for substantial intramolecular heterogeneity in the stable carbon isotopic composition of phytol in photoautotrophic organisms

Author Posting. © Elsevier B.V., 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Organic Geochemistry 39 (2008): 135-146, doi:10.1016/j.orggeochem.2007.09.002.The ubiquitous isoprenoid phytol was isolated from a range of algae, terrestrial plants and a bacterium and its two terminal carbon atoms were quantitatively removed by chemical oxidation. The product, 6,10,14-trimethylpentadecan-2-one, was depleted in 13C by 1-4‰ relative to the parent phytol. This difference is significant, and indicates that the pathway for biosynthesis of phytol induces substantial intramolecular stable carbon isotopic fractionations. The nature and magnitude of the fractionations suggest strongly that it is associated both with the biosynthesis of isopentenyl pyrophosphate via the 2-C-methylerythritol-4-phosphate pathway and with the formation of carotenoids and phytol from geranyl-geraniolphosphate. As a result of these large, intramolecular isotopic differences, diagenetic products formed by loss of C, such as pristane, may be naturally depleted in 13C by several permil relative to phytane.Shell International Petroleum Maatschappij BV is thanked for financial support for the irm- GC-MS facilit

Structure and Orientation of Transmembrane Peptides in Model Membrane Systems as Studied by Solid-State 2H NMR and Molecular Dynamics

Protein-lipid interactions play an essential role in influencing the structure and activity of membrane proteins. In particular, the tilting properties of transmembrane segments of proteins, which adopt in the most cases an alpha-helical conformation, are very important for the function of proteins and can be influenced by changes in the lipid composition. The study of lipid-protein interactions by using natural membrane proteins in lipid bilayers is however very complicated. Therefore, model systems composed of designed peptides and synthetic lipids are more suitable. The present thesis exemplifies such an approach where both the peptide and lipid composition were systematically varied with the use of the so-called WALP and analogous peptides as mimic of the membrane-spanning parts of proteins in bilayers composed of single-species lipids. A new solid-state 2H NMR approach, called GALA (geometric analysis of labeled alanines) is developed for studying how membrane parameters like the hydrophobic bilayer thickness, the lipid-peptide interfacial interactions and the packing properties of the membrane can influence the tilt and azimuthal orientations, and the dynamics of transmembrane segments of proteins. In chapter 2, it was shown that the tilt angle of WALP23 increases slightly but systematically with increasing hydrophobic mismatch in lipid bilayers of decreasing membrane thickness. Interestingly, the direction in which WALP23 is tilted (i.e. the azimuthal or rotation angle) is constant in all studied PC-bilayers. The GALA approach was also developed to non-oriented samples, which mimic better biological membranes than macroscopically oriented samples, enable better control of environmental conditions such as pH and salt concentration, and allow studies with poorly orientable lipids. In chapter 3, the results show by comparing the properties of WALP23 and of its lysine-flanked analog KALP23 that the nature of anchoring residues influences not only the tilt angle but also especially the azimuthal angle. The use of poly-leucine analogs of WALP23 and KALP23 (WLP23 and KLP23) show that the hydrophobicity of the central part of the peptide neither influences the tilt angle, nor the azimuthal angle. In chapter 4, it was shown that changing the properties of the membrane-water interfacial region by adding the anesthetic molecule 2,2,2-trifluoroethanol to the model membranes affects the orientational behaviour of the model peptides depending on the nature of the interfacial anchoring residues. The alcohol interferes with the interfacial interactions of tryptophan-flanked peptides like WALP23, whereas lysine equivalents are insensitive to its presence. The molecular dynamics simulations presented in chapter 5 help understanding the mechanisms by which WALP23 is interacts with the surrounding lipids. The results emphasize the importance of the secondary structure on the orientation of WALP23 in lipid bilayers. In general, the length of simulations is an important issue for giving a representative view on the lipid-peptide interactions involved. Also, some insight is gained on the interfacial interactions and energetic aspects that influence the tilt and rotation angles and the secondary structure. Chapter 6 summarizes the results presented throughout the thesis and suggests future perspectives for the development of the methodology to membrane proteins and antibiotics

How protein transmembrane segments sense the lipid environment

Integral membrane proteins have central roles in a vast number of vital cellular processes. A structural feature that most membrane proteins have in common is the presence of one or more R-helices with which they traverse the lipid bilayer. Because of the interaction with the surrounding lipids, the organization of these transmembrane helices will be sensitive to lipid properties like lateral packing, hydrophobic thickness, and headgroup charge. The helices may adapt to the lipids in different ways, which in turn can influence the structure and function of the intact membrane protein. In this review, we will focus on how the lipid environment influences two specific properties of transmembrane segments: their lateral association and their tilt with respect to the bilayer normal

Influence of flanking residues on tilt and rotation angles of transmembrane peptides in lipid bilayers. A solid-state2H NMR study

To gain insight into the parameters that determine the arrangement of proteins in membranes, 2H NMR experiments were performed to analyze tilt and rotation angles of membrane-spanning α-helical model peptides upon incorporation in diacylphosphatidylcholine bilayers with varying thickness. The peptides consisted of the sequence acetyl-GW2(LA)8LW2A-NH2 (WALP23) and analogues thereof, in which the interfacial Trp residues were replaced by Lys (KALP23) and/or the hydrophobic sequence was replaced by Leu (WLP23 and KLP23). The peptides were synthesized with a single deuterium-labeled alanine at four different positions along the hydrophobic segment. For all peptides a small but systematic increase in tilt angle was observed upon decreasing the bilayer thickness. However, significantly larger tilt angles were obtained for the Lys-flanked KALP23 than for the Trp-flanked WALP23, suggesting that interfacial anchoring interactions of Trp may inhibit tilting. Increasing the hydrophobicity resulted in an increase in tilt angle for the Trp-flanked analogue only. For all peptides the maximum tilt angle obtained was remarkably small (less than 12°), suggesting that further tilting is inhibited, most likely due to unfavorable packing of lipids around a tilted helix. The results furthermore showed that the direction of tilt is determined almost exclusively by the flanking residues:  Trp- and Lys-flanked peptides were found to have very different rotation angles, which were influenced significantly neither by hydrophobicity of the peptides nor by the extent of hydrophobic mismatch. Finally, very small changes in the side chain angles of the deuterated alanine probes were observed in Trp-flanked peptides, suggesting that these peptides may decrease their hydrophobic length to help them to adapt to thin membranes

On the orientation of a designed transmembrane peptide: toward the right tilt angle?

The orientation of the transmembrane peptide WALP23 under small hydrophobic mismatch has been assessed through long-time-scale molecular dynamics simulations of hundreds of nanoseconds. Each simulation gives systematically large tilt angles (>30°). In addition, the peptide visits various azimuthal rotations that mostly depend on the initial conditions and converge very slowly. In contrast, small tilt angles as well as a well-defined azimuthal rotation were suggested by recent solid-state 2H NMR studies on the same system. To optimally compare our simulations with NMR data, we concatenated the different trajectories in order to increase the sampling. The agreement with 2H NMR quadrupolar splittings is spectacularly better when these latter are back-calculated from the concatenated trajectory than from any individual simulation. From these ensembled-average quadrupolar splittings, we then applied the GALA method as described by Strandberg et al. (Biophys J. 2004, 86, 3709-3721), which basically derives the peptide orientation (tilt and azimuth) from the splittings. We find small tilt angles (6.5°), whereas the real observed tilt in the concatenated trajectory presents a higher value (33.5°). We thus propose that the small tilt angles estimated by the GALA method are the result of averaging effects, provided that the peptide visits many states of different azimuthal rotations. We discuss how to improve the method and suggest some other experiments to confirm this hypothesis. This work also highlights the need to run several and rather long trajectories in order to predict the peptide orientation from computer simulations

How protein transmembrane segments sense the lipid environment

Integral membrane proteins have central roles in a vast number of vital cellular processes. A structural feature that most membrane proteins have in common is the presence of one or more R-helices with which they traverse the lipid bilayer. Because of the interaction with the surrounding lipids, the organization of these transmembrane helices will be sensitive to lipid properties like lateral packing, hydrophobic thickness, and headgroup charge. The helices may adapt to the lipids in different ways, which in turn can influence the structure and function of the intact membrane protein. In this review, we will focus on how the lipid environment influences two specific properties of transmembrane segments: their lateral association and their tilt with respect to the bilayer normal

On the orientation of a designed transmembrane peptide: toward the right tilt angle?

The orientation of the transmembrane peptide WALP23 under small hydrophobic mismatch has been assessed through long-time-scale molecular dynamics simulations of hundreds of nanoseconds. Each simulation gives systematically large tilt angles (>30°). In addition, the peptide visits various azimuthal rotations that mostly depend on the initial conditions and converge very slowly. In contrast, small tilt angles as well as a well-defined azimuthal rotation were suggested by recent solid-state 2H NMR studies on the same system. To optimally compare our simulations with NMR data, we concatenated the different trajectories in order to increase the sampling. The agreement with 2H NMR quadrupolar splittings is spectacularly better when these latter are back-calculated from the concatenated trajectory than from any individual simulation. From these ensembled-average quadrupolar splittings, we then applied the GALA method as described by Strandberg et al. (Biophys J. 2004, 86, 3709-3721), which basically derives the peptide orientation (tilt and azimuth) from the splittings. We find small tilt angles (6.5°), whereas the real observed tilt in the concatenated trajectory presents a higher value (33.5°). We thus propose that the small tilt angles estimated by the GALA method are the result of averaging effects, provided that the peptide visits many states of different azimuthal rotations. We discuss how to improve the method and suggest some other experiments to confirm this hypothesis. This work also highlights the need to run several and rather long trajectories in order to predict the peptide orientation from computer simulations

Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat

Dermcidin encodes the anionic amphiphilic peptide DCD-1L, which displays a broad spectrum of antimicrobial activity under conditions resembling those in human sweat. Here, we have investigated its mode of antimicrobial activity. We found that DCD-1L interacts preferentially with negatively charged bacterial phospholipids with a helix axis that is aligned flat on a lipid bilayer surface. Upon interaction with lipid bilayers DCD-1L forms oligomeric complexes that are stabilized by Zn(2+). DCD-1L is able to form ion channels in the bacterial membrane, and we propose that Zn(2+)-induced self-assembly of DCD-1L upon interaction with bacterial lipid bilayers is a prerequisite for ion channel formation. These data allow us for the first time to propose a molecular model for the antimicrobial mechanism of a naturally processed human anionic peptide that is active under the harsh conditions present in human sweat