Location and interaction of idebenone and mitoquinone in a membrane similar to the inner mitochondrial membrane. Comparison with ubiquinone 10.

Oxygen is essential for aerobic life on earth but it is also the origin of harmful reactive oxygen species (ROS). Ubiquinone is par excellence the endogenous cellular antioxidant, but a very hydrophobic one. Because of that, other molecules have been envisaged, such as idebenone (IDE) and mitoquinone (MTQ), molecules having the same redox active benzoquinone moiety but higher solubility. We have used molecular dynamics to determine the location and interaction of these molecules, both in their oxidized and reduced forms, with membrane lipids in a membrane similar to that of the mitochondria. Both IDE and reduced IDE (IDOL) are situated near the membrane interface, whereas both MTQ and reduced MTQ (MTQOL) locate in a position adjacent to the phospholipid hydrocarbon chains. The quinone moieties of both ubiquinone 10 (UQ10) and reduced UQ10 (UQOL10) in contraposition to the same moieties of IDE, IDOL, MTQ and MTQOL, located near the membrane interphase, whereas the isoprenoid chains remained at the middle of the hydrocarbon chains. These molecules do not aggregate and their functional quinone moieties are located in the membrane at different depths but near the hydrophobic phospholipid chains whereby protecting them from ROS harmful effects.

Phospholipid binding of the dengue virus envelope E protein segment containing the conserved His residue.

Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.

Labyrinthopeptin A2 disrupts raft domains.

Labyrinthopeptins constitute a class of ribosomal synthesized peptides belonging to the type III family of lantibiotics. They exist in different variants and display broad antiviral activities as well as show antiallodynic activity. Although their mechanism of action is not understood, it has been described that Labyrinthopeptins interact with membrane phospholipids modulating its biophysical properties and point out to membrane destabilization as its main point of action. We have used all-atom molecular dynamics to study the location of labyrinthopeptin A2 in a complex membrane as well as the existence of specific interactions with membrane lipids. Our results indicate that labyrinthopeptin A2, maintaining its globular structure, tends to be placed at the membrane interface, mainly between the phosphate atoms of the phospholipids and the oxygen atom of cholesterol modulating the biophysical properties of the membrane lipids. Outstandingly, we have found that labyrinthopeptin A2 tends to be preferentially surrounded by sphingomyelin while excluding cholesterol. The bioactive properties of labyrinthopeptin A2 could be attributed to the specific disorganization of raft domains in the membrane and the concomitant disruption of the overall membrane organization. These results support the improvement of Labyrinthopeptins as therapeutic molecules, opening up new opportunities for future medical advances.

SARS-CoV-2 Protein S Fusion Peptide Is Capable of Wrapping Negatively-Charged Phospholipids.

COVID-19, caused by SARS-CoV-2, which is a positive-sense, single-stranded RNA enveloped virus, emerged in late 2019 and was declared a worldwide pandemic in early 2020 causing more than 600 million infections so far and more than 6 million deaths in the world. Although new vaccines have been implemented, the pandemic continues to impact world health dramatically. Membrane fusion, critical for the viral entry into the host cell, is one of the main targets for the development of novel antiviral therapies to combat COVID-19. The S2 subunit of the viral S protein, a class I membrane fusion protein, contains the fusion domain which is directly implicated in the fusion mechanism. The knowledge of the membrane fusion mechanism at the molecular level will undoubtedly result in the development of effective antiviral strategies. We have used all-atom molecular dynamics to analyse the binding of the SARS-CoV-2 fusion peptide to specific phospholipids in model membranes composed of only one phospholipid plus cholesterol in the presence of either Na+ or Ca2+. Our results show that the fusion peptide is capable of binding to the membrane, that its secondary structure does not change significantly upon binding, that it tends to preferentially bind electronegatively charged phospholipids, and that it does not bind cholesterol at all. Understanding the intricacies of the membrane fusion mechanism and the molecular interactions involved will lead us to the development of antiviral molecules that will allow a more efficient battle against these viruses.

Bergamottin: location, aggregation and interaction with the plasma membrane.

Bioactive furanocoumarins, a group of natural secondary metabolites common in higher plants, are recognized for their benefits to human health and have been shown to have numerous biological properties. However, the knowledge of its biomolecular mechanism is not known. One of the main furanocoumarins is bergamottin (BGM), which is characterized by a planar three-ringed structure and a hydrocarbon chain, which give BGM its high lipid/water partition coefficient. Because of that, and although the biological mechanism of BGM is not known, BGM bioactive properties could be ascribed to its potential to interact with the biological membrane, modulating its structure, changing its dynamics and at the same time that it might interact with lipids. For our goal, we have applied molecular dynamics to determine the position of BGM in a complex membrane and discern the possibility of certain interactions with membrane lipids. Our findings establish that BGM tends to locate in the middle of the hydrocarbon layer of the membrane, inserts in between the hydrocarbon chains of the phospholipids in an oblique position with respect to the membrane plane, increasing the fluidity of the membrane. Significantly, BGM tends to be surrounded by POPC molecules but exclude the molecule of CHOL. Outstandingly, BGM molecules associate spontaneously creating aggregates, which does not preclude them from interacting with and inserting into the membrane. The bioactive properties of BGM could be ascribed to its membranotropic effects and support the improvement of these molecules as therapeutic molecules, giving place to new opportunities for potential medical improvements.Communicated by Ramaswamy H. Sarma.

Interaction of Lassa virus fusion and membrane proximal peptides with late endosomal membranes.

Mammarenaviruses include many significant worldwide-widespread human pathogens, among them Lassa virus (LASV), having a dramatic morbidity and mortality rate. They are a potential high-risk menace to the worldwide public health since there are no treatments and there is a high possibility of animal-to-human and human-to-human viral transmission. These viruses enter into the cells by endocytosis fusing its membrane envelope with the late endosomal membrane thanks to the glycoprotein GP2, a membrane fusion protein of class I. This protein contains different domains, among them the N-terminal fusion peptide (NFP), the internal fusion loop (IFL), the membrane proximal external region (MPER) and the transmembrane domain (TMD). All these domains are implicated in the membrane fusion process. In this work, we have used an all-atom molecular dynamics study to know the binding of these protein domains with a complex membrane mimicking the late endosome one. We show that the NFP/IFL domain is capable of spontaneously inserting into the membrane without a significant change of secondary structure, the MPER domain locates at the bilayer interface with an orientation parallel to the membrane surface and tends to interact with other MPER domains, and the TMD domain tilts inside the bilayer. Moreover, they predominantly interact with negatively charged phospholipids. Overall, these membrane-interacting domains would characterise a target that would make possible to find effective antiviral molecules against LASV in particular and Mammarenaviruses in general.

Procyanidin C1 Location, Interaction, and Aggregation in Two Complex Biomembranes.

Procyanidins are known for their many benefits to human health and show a plethora of biological effects. One of the most important procyanidin is the procyanidin trimer C1 (PC1). Due to its relatively high lipid-water partition coefficient, the properties of PC1 could be attributed to its capability to interact with the biomembrane, to modulate its structure and dynamics, and to interact with lipids and proteins, however, its biological mechanism is not known. We have used all-atom molecular dynamics in order to determine the position of PC1 in complex membranes and the presence of its specific interactions with membrane lipids, having simulated a membrane mimicking the plasma membrane and another mimicking the mitochondrial membrane. PC1 has a tendency to be located at the membrane interphase, with part of the molecule exposed to the water solvent and part of it reaching the first carbons of the hydrocarbon chains. It has no preferred orientation, and it completely excludes the CHOL molecule. Remarkably, PC1 has a tendency to spontaneously aggregate, forming high-order oligomers. These data suggest that its bioactive properties could be attributed to its membranotropic effects, which therefore supports the development of these molecules as therapeutic molecules, which would open new opportunities for future medical advances.

Envelope E protein of dengue virus and phospholipid binding to the late endosomal membrane.

Flaviviruses include many significant human pathogens, comprising dengue, West Nile, Yellow fever, Japanese encephalitis, Zika and tick-borne encephalitis viruses and many others, affecting millions of people in the world. These viruses have produced important epidemics in the past, they continue to do it and they will undoubtedly continue to do so in the future. Flaviviruses enter into the cells via receptor-mediated endocytosis by fusing its membrane with the endosomal membrane in a pH-dependent manner with the help of the envelope E protein, a prototypical class II membrane fusion protein. The envelope E protein has a conserved fusion peptide at its distal end, which is responsible in the first instance of inserting the protein into the host membrane. Since the participation of other segments of the E protein in the fusion process should not be ruled out, we have used atomistic molecular dynamics to study the binding of the distal end of domain II of the envelope E protein from Dengue virus (DENV) with a complex membrane similar to the late-endosome one. Our work shows that not only the fusion peptide participates directly in the fusion, but also two other sequences of the protein, next to the fusion peptide it in the three-dimensional structure, are jointly wrapped in the fusion process. Overall, these three sequences represent a new target that would make it possible to obtain effective antivirals against DENV in particular and Flaviviruses in general.

Aggregation of 25-hydroxycholesterol in a complex biomembrane. Differences with cholesterol.

25-Hydroxycholesterol (25HC), one of the most important oxysterol molecules, can be used by cells to fight bacterial and viral infections but the mechanism that defines its biological effects are unknown. Using molecular dynamics, we have aimed to describe the orientation and location of 25HC in the membrane as well as the interactions it might have with lipids. We have studied two complex model membrane systems, one similar to the late endosome membrane and the other one to the plasma membrane. Our results reinforce that 25HC is inserted in the membrane in a relative stable location similar to but not identical to cholesterol. 25HC fluctuates in the membrane to a much greater degree than cholesterol, but the effect of 25HC on the phospholipid order parameters is not significantly different. One of the most notable facts about 25HC is that, unlike cholesterol, this molecule tends to aggregate, forming dimers, trimers and higher-order aggregates. These aggregates are formed spontaneously through the formation of hydrogen bonds between the two 25HC atoms, the formation of hydrogen bonds being independent of the studied system. Remarkably, no contacts or hydrogen bonds are observed between 25HC and cholesterol molecules, as well as between cholesterol molecules themselves at any time. It would be conceivable that 25HC, by forming high order aggregates without significantly altering the membrane properties, would modify the way proteins interact with the membrane and henceforth form a true innate antiviral molecule.

Location, Orientation and Aggregation of Bardoxolone-ME, CDDO-ME, in a Complex Phospholipid Bilayer Membrane.

Bardoxolone methyl (CDDO-Me), a synthetic derivative of the naturally occurring triterpenoid oleanolic acid, displays strong antioxidant, anticancer and anti-inflammatory activities, according to different bibliographical sources. However, the understanding of its molecular mechanism is missing. Furthermore, CDDO-Me has displayed a significant cytotoxicity against various types of cancer cells. CDDO-Me has a noticeable hydrophobic character and several of its effects could be attributed to its ability to be incorporated inside the biological membrane and therefore modify its structure and specifically interact with its components. In this study, we have used full-atom molecular dynamics to determine the location, orientation and interactions of CDDO-Me in phospholipid model membranes. Our results support the location of CDDO-Me in the middle of the membrane, it specifically orients so that the cyano group lean towards the phospholipid interface and it specifically interacts with particular phospholipids. Significantly, in the membrane the CDDO-Me molecules specifically interact with POPE and POPS. Moreover, CDDO-Me does not aggregates in the membrane but it forms a complex conglomerate in solution. The formation of a complex aggregate in solution might hamper its biological activity and therefore it should be taken into account when intended to be used in clinical assays. This work should aid in the development of these molecules opening new avenues for future therapeutic developments.

Identification of the phospholipid binding regions of the envelope E protein of flaviviruses by molecular dynamics.

The Flavivirus genus comprise several important human pathogens, including dengue, West Nile, Yellow fever, Japanese encephalitis, Zika, and tick-borne encephalitis viruses. These enveloped viruses affect more than 2 billion people in the world, mainly in less developed countries. Although some vaccines exist for some flaviviruses, these vaccines are not universally available due to many factors and since their infections are a world-wide public health issue, the development of antiviral molecules is fundamental. Flavivirus membranes, through the help of the envelope E glycoprotein, fuse with endosomal compartments in a pH-dependent way to release their genome into the cytoplasm and require specific lipids, such as bis(monoacylglycero)phosphate (BMP), for efficient fusion. The fundamental role the envelope E protein has on viral entry and membrane fusion suggest that it is an essential antiviral target. In this work, we have used atomistic molecular dynamics simulations to study the binding of the head-group of BMP to the tip of the envelope E proteins of ZIKV, DENV, TBEV and JEV viruses whose three-dimensional structures are known. Our results indicate that, apart from the fusion loop, there are different amino acid residues in different regions of the envelope E proteins of flaviviruses capable of binding the head-group of BMP. These regions should work together to accomplish the binding and fusion of the envelope and endosomal membranes and represent a new target to develop and design potent and effective antiviral agents capable of blocking flavivirus-endosome membrane fusion. [Formula: see text].

Epigallocatechin-3-gallate location and interaction with late endosomal and plasma membrane model membranes by molecular dynamics.

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea and it has been reported to have many beneficial properties against many different types of illnesses and infections. However, the exact mechanism/s underlying its biological effects are unknown. It has been previously shown that EGCG is capable of binding to and disrupting the membrane, so that some of its effects on biological systems could be ascribed to its capacity to incorporate into the biological membrane and modulate its structure. In this work, we have used atomistic molecular dynamics (MD) to discern the location and orientation of EGCG in model membranes and the possible existence of specific interactions with membrane lipids. For that goal, we have used in our simulation two complex model membranes, one resembling the plasma membrane (PM) and the other one the late endosome (LE) membrane. Our results support that EGCG tends to associate with the membrane and exists inside it in a relatively stable and steady location with a low propensity to be associated with other EGCG molecules. Interestingly, EGCG forms hydrogen bonds with POPC and POPE in the PM system but POPC and BMP and no POPE in the LE. These data suggest that the broad beneficial effects of EGCG could be mediated, at least in part, through its membranotropic effects and therefore membrane functioning. Communicated by Ramaswamy H. Sarma.

Spontaneous membrane insertion of a dengue virus NS2A peptide.

Non-structural NS2A protein of Dengue virus is essential for viral replication but poorly characterized because of its high hydrophobicity. We have previously shown experimentally that NS2A possess a segment, peptide dens25, known to insert into membranes and interact specifically with negatively-charged phospholipids. To characterize its membrane interaction we have used two types of molecular dynamics membrane model systems, a highly mobile membrane mimetic (HMMM) and an endoplasmic reticulum (ER) membrane-like model. Using the HMMM system, we have been able of demonstrating the spontaneous binding of dens25 to the negatively-charged phospholipid 1,2-divaleryl-sn-glycero-3-phosphate containing membrane whereas no binding was observed for the membrane containing the zwitterionic one 1,2-divaleryl-sn-glycero-3-phosphocholine. Using the ER-like membrane model system, we demonstrate the spontaneous insertion of dens25 into the middle of the membrane, it maintained its three-dimensional structure and presented a nearly parallel orientation with respect to the membrane surface. Both charged and hydrophobic amino acids, presenting an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment, are responsible for membrane binding and insertion. Dens25 might control protein/membrane interaction and be involved in membrane rearrangements critical for the viral cycle. These data should help us in the development of inhibitor molecules that target NS2A segments involved in membrane reorganisation.

Location of the bioactive pentacyclic triterpene ursolic acid in the membrane. A molecular dynamics study.

Ursolic acid (URS), an ursane-representative bioactive pentacyclic triterpene, is a plant secondary metabolite presenting a great number of pharmacological beneficial properties. Due to the prominent hydrophobic character of URS and its high phospholipid/water partition coefficient, some of its possible effects on biological systems might be related to its capacity to interact with and locate into the membrane as well as interact specifically with its components. In this work, we have studied the location and orientation of URS in the membrane by molecular dynamics simulations. At the end of the simulation, URS locates near the surface in vicinity to the phospholipid headgroups but its orientation depends on lipid composition, its final average orientation being a nearly parallel one in POPC but a nearly perpendicular one in POPC/POPE/POPG/PSM/Chol. Furthermore, in the complex lipid system URS seems to interact specifically with POPE, PSM, and Chol excluding POPG from its surroundings, which could lead to phase separation and domain formation. The different disposition of URS in the membrane and its specific interaction with certain lipid types could lead to a significant perturbation of the membrane structure. The important pharmacological activities of URS would rely on the effects it exerts on the membrane structure in general and the existence of specific interactions with specific lipids in particular.

Molecular dynamics study of the membrane interaction of a membranotropic dengue virus C protein-derived peptide.

Dengue virus C protein, essential in the dengue virus life cycle, possesses a segment, peptide PepC, known to bind membranes composed of negatively charged phospholipids. To characterize its interaction with the membrane, we have used the molecular dynamics HMMM membrane model system. This approach is capable of achieving a stable system and sampling the peptide/lipid interactions which determine the orientation and insertion of the peptide upon membrane binding. We have been able to demonstrate spontaneous binding of PepC to the 1,2-divaleryl-sn-glycero-3-phosphate/1,2-divaleryl-sn-glycero-3-phosphocholine membrane model system, whereas no binding was observed at all for the 1,2-divaleryl-sn-glycero-3-phosphocholine one. PepC, adopting an α-helix profile, did not insert into the membrane but did bind to its surface through a charge anchor formed by its three positively charged residues. PepC, maintaining its three-dimensional structure along the whole simulation, presented a nearly parallel orientation with respect to the membrane when bound to it. The positively charged amino acid residues Arg-2, Lys-6, and Arg-16 are mainly responsible for the peptide binding to the membrane stabilizing the structure of the bound peptide. The segment of dengue virus C protein where PepC resides is a fundamental protein-membrane interface which might control protein/membrane interaction, and its positive amino acids are responsible for membrane binding defining its specific location in the bound state. These data should help in our understanding of the molecular mechanism of DENV life cycle as well as making possible the future development of potent inhibitor molecules, which target dengue virus C protein structures involved in membrane binding.

The Location of the Protonated and Unprotonated Forms of Arbidol in the Membrane: A Molecular Dynamics Study.

Arbidol is a potent broad-spectrum antiviral molecule for the treatment and prophylaxis of many viral infections. Viruses that can be inhibited by arbidol include enveloped and non-enveloped viruses, RNA and DNA viruses, as well as pH-independent and pH-dependent ones. These differences in viral types highlight the broad spectrum of Arb antiviral activity and, therefore, it must affect a common viral critical step. Arbidol incorporates rapidly into biological membranes, and some of its antiviral effects might be related to its capacity to interact with and locate into the membrane. However, no information is available of the molecular basis of its antiviral mechanism/s. We have aimed to locate the protonated (Arp) and unprotonated (Arb) forms of arbidol in a model membrane system. Both Arb and Arp locate in between the hydrocarbon acyl chains of the phospholipids but its specific location and molecular interactions differ from each other. Whereas both Arb and Arp average location in the membrane palisade is a similar one, Arb tends to be perpendicular to the membrane surface, whereas Arp tends to be parallel to it. Furthermore, Arp, in contrast to Arb, seems to interact stronger with POPG than with POPC, implying the existence of a specific interaction between Arp, the protonated from, with negatively charged phospholipids. This data would suggest that the active molecule of arbidol in the membrane is the protonated one, i.e., the positively charged molecule. The broad antiviral activity of arbidol would be defined by the perturbation it exerts on membrane structure and therefore membrane functioning.

Oleuropein aglycone in lipid bilayer membranes. A molecular dynamics study.

Olive oil has been recognized to possess many therapeutic applications. Its beneficial effects arise from many causes, but one of them lies on the presence of oleuropein aglycone (OA). OA presents a plethora of pharmacological beneficial properties. Although there is a great research going on the effect of polyphenols and their derivatives on different aspects of health, much less knowledge is available of the molecular basis of their beneficial effects. Due to the prominent hydrophobic character of OA and its high phospholipid/water partition coefficient, some of its possible effects on biological systems might be related to its capacity to interact with and locate into the membrane. In this work we have aimed to locate the molecule of OA in two membrane model systems, i.e., POPC/Chol and POPC/POPG/Chol. OA locates in between the hydrocarbon acyl chains of the phospholipids but its specific location and molecular interactions differ depending on the lipid system. OA is nearer to the membrane surface in the POPC/Chol system but it is located at a deeper position in the POPC/POPG/Chol system. Furthermore, OA seems to interact stronger with POPG than with POPC, implying the existence of specific interactions with negatively-charged phospholipids. Some of the biological effects of OA could be due to its preferential location in the membrane depending on the membrane lipid composition as well as the existence of specific interactions with specific phospholipids.

Membrane interacting regions of Dengue virus NS2A protein.

The Dengue virus (DENV) NS2A protein, essential for viral replication, is a poorly characterized membrane protein. NS2A displays both protein/protein and membrane/protein interactions, yet neither its functions in the viral cycle nor its active regions are known with certainty. To highlight the different membrane-active regions of NS2A, we characterized the effects of peptides derived from a peptide library encompassing this protein's full length on different membranes by measuring their membrane leakage induction and modulation of lipid phase behavior. Following this initial screening, one region, peptide dens25, had interesting effects on membranes; therefore, we sought to thoroughly characterize this region's interaction with membranes. This peptide presents an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment. We show that dens25 strongly interacts with membranes that contain a large proportion of lipid molecules with a formal negative charge, and that this effect has a major electrostatic contribution. Considering its membrane modulating capabilities, this region might be involved in membrane rearrangements and thus be important for the viral cycle.

Membranotropic regions of the dengue virus prM protein.

The Dengue virus (DENV) prM protein consists of two moieties, the pr and M domains. Apart from preventing the premature fusion activity of the DENV E protein, prM has several other unknown biological roles, displaying both protein-protein and membrane-protein interactions. Although the prM protein is an essential component of the DENV viral cycle, little is known about its biological functions and what regions of this protein are responsible for said functions. By performing an exhaustive study of membrane rupture induced by a prM peptide library on simple and complex model membranes as well as their ability to modulate the phospholipid phase transitions of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol], we identified six membranotropic regions on the prM protein. Apart from the previously identified two transmembrane segments of the protein, one of these regions probably interacts with the fusion E protein and another one, the stem segment, would interact with the membrane modulating its structure. These data will help us understand the molecular mechanism of viral entry and morphogenesis, allow the identification of new targets for the treatment of Dengue virus infection, and make possible the future development of DENV entry inhibitors.

Electrostatically driven lipid-lysozyme mixed fibers display a multilamellar structure without amyloid features.

Understanding the interactions between anionic lipid membranes and amyloidogenic proteins/peptides is key to elucidate the molecular mechanisms underlying the membrane-driven amyloid fiber formation. Here, hen egg-white lysozyme was used as a model protein to test whether this same process also occurs with non-amyloidogenic lipid-binding proteins/peptides. A complementary set of biophysical techniques was employed to study the structure and dynamics of the lipid-lysozyme mixed fibers produced at a low lipid/protein molar ratio that have been proposed earlier to present "amyloid-like" characteristics. The multilamellar architecture of these elongated mesoscopic structures was established by performing time-resolved Förster resonance energy transfer measurements, at both bulk (ensemble) and single-fiber level. The predominantly oligomeric lysozyme and phospholipids were both found to display significantly decreased lateral mobility when embedded in these mixed fibers. Notably, two-photon microscopy of Laurdan revealed that a pronounced membrane surface dehydration/increased molecular interfacial packing was produced exclusively in these elongated mixed supramolecular fibers present in the highly polymorphic samples. Infrared spectroscopic studies of lysozyme in these samples further showed that this protein did not exhibit a rich ß-sheet structure characteristic of amyloid fibrils. These results support the conclusion that negatively charged lipid membranes do not have the general ability to trigger amyloid fibril formation of non-amyloidogenic proteins.