Thomas James McIntosh

The overall goal of our research is to determine how lipid-mediated forces modulate conformation, binding, and distribution of biologically significant interfacial and transmembrane peptides. In the past year we have been working on two separate projects: (1) the mechanisms by which proteins and lipids are concentrated in specialized regions of the membrane and (2) the role of membrane components in governing the lytic effects of antimicrobial peptides.
Specific proteins and lipids are sequestered to regions of cell membranes called microdomains or “rafts”. Due to their ability to sequester and concentrate these lipids and proteins, rafts are thought to play important roles in many physiological processes, such as signal transduction, membrane fusion, and protein trafficking. We have been analyzing bilayers composed of unsaturated phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol that have similar detergent insolubility characteristics as rafts isolated from cell plasma membranes. We have chemically characterized the fractions corresponding to detergent soluble membranes (DSMs) and detergent resistant membranes (DRMs, thought to be equivalent to rafts) from 1:1:1 PC:SM:cholesterol, compared the binding properties of selected peptides to bilayers with the compositions of DSMs and DRMs, and determined the structure of DRMs with X-ray diffraction. Compared to the equimolar starting material, DRMs were enriched in both SM and cholesterol. Both transmembrane and interfacial peptides bound to a greater extent to DSM bilayers than to DRM bilayers, likely because of differences in the mechanical properties of the two bilayers. Due to their high content of sphingomyelin and cholesterol, raft bilayers were 10 Å thicker than non-raft bilayers. It has been postulated that rafts concentrate proteins with long transbilayer domains because of “hydrophobic matching” between the transbilayer domain and the thick bilayer hydrocarbon region. However, because the area compressibility modulus of SM:cholesterol bilayers is larger than that of non-raft bilayers, there should be an energy cost to partition proteins or peptides into rafts. To determine the effects on peptide sorting of raft thickness and mechanical properties, we incorporated two transbilayer peptides (P-23, P-29) into 1:1:1 PC:SM:cholesterol bilayers and then extracted DSMs and DRMs. P-23 and P-29 were designed to have transbilayer domains that matched the hydrocarbon thicknesses of DSMs and DRMs, respectively. At 4 oC, the temperature at which most both detergent extractions are performed, both P-23 and P-29 preferentially localized to DSMs. The free energy barrier to move either P-23 (1.3 kcal/mol) or P-29 (1.0 kcal/mol) to DRMs was comparable to that required to move PC (1.0 kcal/mol). However, at 37 oC more P-29 than P-23 was located in DRMs, implying that hydrophobic matching played a larger role in peptide sorting at 37 oC than at 4 oC, probably due to temperature-dependent differences in raft mechanical properties. These experiments indicate that: (1) both bilayer mechanical properties and hydrophobic matching impact peptide distribution between DSMs and DRMs and (2) detergent extraction at 4 oC does not give a precise measurement of the distribution of transmembrane proteins at physiological temperature.
In terms of our second project, a variety of organisms, from insects to humans, deploy a chemical barrier at the first possible point of contact with microbes. This line of defense depends largely on small, secreted peptides that are effective against a wide range of microbial invaders. However, the sensitivity to antimicrobial peptides varies among cell types, so that host cells are insensitive to the peptides and Gram-positive bacteria are in general more sensitive than Gram-negative bacteria. We have been initially studying melittin, a small, cationic peptide that, like many other antimicrobial peptides, lyses cell membranes by acting on their lipid bilayers. We have performed direct binding and vesicle leakage experiments to determine the sensitivity to melittin of bilayers composed of various physiologically relevant lipids, such as key components of the cell membranes of eukaryotic cells (cholesterol and PC) and Gram-positive bacteria (bacterial phospholipids), and the outer membranes of Gram-negative bacteria (lipopolysaccharide or LPS). Melittin binds to bilayers composed of both zwitterionic and negatively charged phospholipids, as well as to the highly charged LPS bilayers. The magnitude of the free energy of binding increased with increasing bilayer charge density, from -7.6 kcal/mol for PC bilayers to -8.9 to –11.0 kcal/mol for negatively charged bilayers, such as bilayers containing phospholipids with covalently attached polyethyleneglycol (PEG-lipids) or LPS bilayers. Comparisons of these data showed that binding was not markedly affected by the steric barrier produced by the PEG in PEG-lipids or by the polysaccharide core of LPS. The addition of equimolar cholesterol to PC bilayers reduced binding and reduced the melittin-induced leakage by 20-fold. Moreover, LPS had a similar high resistance to melittin-induced leakage as 1:1 PC:cholesterol bilayers. These results indicate that cholesterol in eukaryotic plasma membranes and LPS in Gram-negative bacteria provide strong protection against the lytic effects of melittin, explaining the resistance to antimicrobial peptides of these cell types. We argue that this resistance is due at least in part to the similar tight packing of the lipid acyl chains in PC:cholesterol and LPS bilayers.