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Mitochondrial dysfunction underlies many heritable diseases, acquired pathologies, and aging-related declines in health. Szeto–Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that are efficacious toward a wide array of mitochondrial disorders and are believed to target mitochondrial membranes because they are enriched in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, using biophysical and computational approaches, we have analyzed the interactions of the lead compound SS-31 (elamipretide) with model and mitochondrial membranes. Our results show that this polybasic peptide partitions into the membrane interfacial region with an affinity and a lipid binding density that are directly related to surface charge. We found that SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it did cause saturable alterations in lipid packing. Most notably, SS-31 modulated the surface electrostatics of both model and mitochondrial membranes. We propose nonexclusive mechanisms by which the tuning of surface charge could underpin the mitoprotective properties of SS-31, including alteration of the distribution of ions and basic proteins at the interface, and/or modulation of bilayer physical properties. As a proof of concept, we show that SS-31 alters divalent cation (calcium) distribution within the interfacial region and reduces the energetic burden of calcium stress in mitochondria. The mechanistic details of SS-31 revealed in this study will help inform the development of future compound variants with enhanced efficacy and bioavailability.

This is a perspective article entitled “Frontiers in computational biophysics: understanding conformational dynamics of complex lipid mixtures relevant to biology” which is following a CECAM meeting with the same name. Fil: Friedman, Ran. Linnæus University; Argentina Fil: Khalid, Syma. University of Southampton; Reino Unido Fil: Aponte Santamaría, Camilo. Ruprecht-Karls-Universität Heidelberg; Alemania. Universidad de los Andes; Colombia Fil: Arutyunova, Elena. University of Alberta; Canadá Fil: Becker, Marlon. Westfälische Wilhelms Universität; Alemania Fil: Boyd, Kevin J.. University of Connecticut; Estados Unidos Fil: Christensen, Mikkel. University Aarhus; Dinamarca Fil: Coimbra, João T. S.. Universidad de Porto; Portugal Fil: Concilio, Simona. Universita di Salerno; Italia Fil: Daday, Csaba. Heidelberg Institute for Theoretical Studies; Alemania Fil: Eerden, Floris J. van. University of Groningen; Países Bajos Fil: Fernandes, Pedro A.. Universidad de Porto; Portugal Fil: Gräter, Frauke. Heidelberg University; Alemania. Heidelberg Institute for Theoretical Studies; Alemania Fil: Hakobyan, Davit. Westfälische Wilhelms Universität; Alemania Fil: Heuer, Andreas. Westfälische Wilhelms Universität; Alemania Fil: Karathanou, Konstantina. Freie Universität Berlin; Alemania Fil: Keller, Fabian. Westfälische Wilhelms Universität; Alemania Fil: Lemieux, M. Joanne. University of Alberta; Canadá Fil: Marrink, Siewert J.. University of Groningen; Países Bajos Fil: May, Eric R.. University of Connecticut; Estados Unidos Fil: Mazumdar, Antara. University of Groningen; Países Bajos Fil: Naftalin, Richard. Colegio Universitario de Londres; Reino Unido Fil: Pickholz, Mónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Piotto, Stefano. Universita di Salerno; Italia Fil: Pohl, Peter. Johannes Kepler University; Austria Fil: Quinn, Peter. Colegio Universitario de Londres; Reino Unido Fil: Ramos, Maria J.. Universidad de Porto; Portugal Fil: Schiøtt, Birgit. University Aarhus; Dinamarca Fil: Sengupta, Durba. National Chemical Laboratory India; India Fil: Sessa, Lucia. Universita di Salerno; Italia Fil: Vanni, Stefano. University Of Fribourg; Fil: Zeppelin, Talia. University Aarhus; Dinamarca Fil: Zoni, Valeria. University of Fribourg; Suiza Fil: Bondar, Ana-Nicoleta. Freie Universität Berlin; Alemania Fil: Domene, Carmen. University of Oxford; Reino Unido. University of Bath; Reino Unido

Mitochondrial dysfunction includes heritable diseases, acquired pathologies, and age-related declines in health. Szeto-Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that have demonstrated efficacy in treating a wide array of mitochondrial disorders, and are believed to target mitochondrial membranes due to their enrichment in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, we have analyzed the interactions of the lead compound SS-31 (Elamipretide) with model and mitochondrial membranes using biophysical and computational approaches. Our results show that this polybasic peptide partitions into the membrane interfacial region with affinity and binding density that are directly related to surface charge. SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it does cause saturable alterations in lipid packing. Most notably, SS-31 modulates the surface electrostatic properties of model and mitochondrial membranes, which could play a significant role in the mitoprotective properties of this compound. As a proof of concept, we show that SS-31 alters ion distribution at the membrane interface with implications for maintaining mitochondrial membranes subject to divalent cation (calcium) stress. Taken together, these results support a mechanism of action in which SS peptides interact with lipid bilayers and alter the biophysical (primarily electrostatic) properties of mitochondrial membranes as their primary mechanism of action. Understanding this molecular mechanism is key to the development of future compound variants with enhanced efficacy.SignificanceSzeto-Schiller (SS) peptides are among the most promising therapeutic compounds for mitochondrial dysfunction. However, the molecular target(s) and the mechanism of action of SS peptides are poorly understood. In this study, we evaluate the interaction of the lead compound SS-31 (Elamipretide) with mitochondrial and synthetic model membranes using a host of biophysical techniques. Our results show that SS-31 membrane interaction is driven largely by the negative surface charge of mitochondrial membranes and that SS-31 alters lipid bilayer properties, most notably electrostatics at the membrane interface. This work supports a mechanism in which SS peptides act on a key physical property of mitochondrial membranes rather than with a specific protein complex, consistent with the exceptionally broad therapeutic efficacy of these compounds.

Flock House virus (FHV) serves as a model system for understanding infection mechanisms utilized by non-enveloped viruses to transport across cellular membranes. During the infection cycle of FHV, a fundamental stage involves disruption of the endosomal membrane by membrane active peptides, following externalization of the peptides from the capsid interior. The FHV lytic agents are the 44 C-terminal amino acids residues of the capsid protein, which are auto-catalytically cleaved during the capsid maturation process. The cleaved peptides are termed γ peptides. In this study, we perform multi-scale molecular dynamics simulations including 40 μs all-atom molecular dynamics simulations to study the behavior of pre-inserted transmembrane lytic peptides at a high concentration in a neutral membrane. We study the dynamical organization among peptides to form oligomeric bundles in four systems including the wild-type γ peptide and three mutant forms; namely, a truncation mutant in which the 23 C-terminal residues are deleted (γ1), a construct where the 8 C-terminal residues of γ are fused to γ1 (Δ385–399 γ) and a single-point mutant (F402A γ), all of which have been experimentally shown to drastically affect infectivity and lytic activity compared to the wild-type γ. Our results shed light on the actions of varied forms of the FHV lytic peptide including membrane insertion, trans-membrane stability, peptide oligomerization, water permeation activity and dynamic pore formation. Findings from this study provide detailed structural information and rationale for the differences in lytic activity among variants of FHV γ.

Molecular dynamics is a powerful tool to investigate atomistic and mesoscopic phenomena in lipid bilayer systems. These studies have progressed with the advent of increased computational power, and efforts are now increasingly being directed toward investigating the role of curvature and bilayer morphology, as these are critical features of biological processes. Computational studies of lipid bilayers benefit from tools that can create starting configurations for molecular dynamics simulations, but the majority of such tools are restricted to generating flat bilayers. Generating curved bilayer configurations comes with practical complications and potential ramifications on physical properties in the simulated system if the bilayer is initiated in a high-strain state. We present a new tool for creating curved lipid bilayers that combines flexibility of shape, force field, model resolution, and bilayer composition. A key aspect of our approach is the use of the monolayer pivotal plane location to accurately estimate interleaflet area differences in a curved bilayer. Our tool is named BUMPy (Building Unique Membranes in Python), is written in Python, is fast, and has a simple command line interface.

The mitochondrial lipid cardiolipin (CL) contributes to the spatial protein organization and morphological character of the inner mitochondrial membrane. Monolysocardiolipin (MLCL), an intermediate species in the CL remodeling pathway, is enriched in the multisystem disease Barth syndrome. Despite the medical relevance of MLCL, a detailed molecular description that elucidates the structural and dynamic differences between CL and MLCL has not been conducted. To this end, we performed comparative atomistic molecular dynamics studies on bilayers consisting of pure CL or MLCL to elucidate similarities and differences in their molecular and bulk bilayer properties. We describe differential headgroup dynamics and hydrogen bonding patterns between the CL variants and show an increased cohesiveness of MLCL's solvent interfacial region, which may have implications for protein interactions. Finally, using the coarse-grained Martini model, we show that substitution of MLCL for CL in bilayers mimicking mitochondrial composition induces drastic differences in bilayer mechanical properties and curvature-dependent partitioning behavior. Together, the results of this work reveal differences between CL and MLCL at the molecular and mesoscopic levels that may underpin the pathomechanisms of defects in cardiolipin remodeling.

Mitochondrial metabolic function is affected by the morphology and protein organization of the mitochondrial inner membrane. Cardiolipin (CL) is a unique tetra-acyl lipid that is involved in the maintenance of the highly curved shape of the mitochondrial inner membrane as well as spatial organization of the proteins necessary for respiration and oxidative phosphorylation. Cardiolipin has been suggested to self-organize into lipid domains due to its inverted conical molecular geometry, though the driving forces for this organization are not fully understood. In this work, we use coarse-grained molecular dynamics simulations to study the mechanical properties and lipid dynamics in heterogeneous bilayers both with and without CL, as a function of membrane curvature. We find that incorporation of CL increases bilayer deformability and that CL becomes highly enriched in regions of high negative curvature. We further show that another mitochondrial inverted conical lipid, phosphatidylethanolamine (PE), does not partition or increase the deformability of the membrane in a significant manner. Therefore, CL appears to possess some unique characteristics that cannot be inferred simply from molecular geometry considerations.

The TIM23 complex of the mitochondrial inner membrane mediates the translocation and integration of most nuclear-encoded polypeptides that reside within the mitochondrion. The central subunit of this complex is Tim23, a voltage-gated channel-forming protein that constitutes part of the polypeptide-conducting channel. Tim23 has a bipartite domain organization with a carboxy-terminal integral membrane domain and an amino-terminal intrinsically disordered region that resides in the intermembrane space (IMS).

Norwalk virus causes severe gastroenteritis for which there is currently no specific anti-viral therapy. A stage of the infection process is uncoating of the protein capsid to expose the viral genome and allow for viral replication. A mechanical characterization of the Norwalk virus may provide important information relating to the mechanism of uncoating. The mechanical strength of the Norwalk virus has previously been investigated using atomic force microscopy (AFM) nanoindentation experiments. Those experiments cannot resolve specific molecular interactions, and therefore, we have employed a molecular modeling approach to gain insights into the potential uncoating mechanism of the Norwalk capsid. In this study, we perform simulated nanoindentation using a coarse-grained structure-based model, which provides an estimate of the spring constant in good agreement with the experimentally determined value. We further analyze the fracture mechanisms and determine weak interfaces in the capsid structure, which are potential sites to inhibit uncoating by stabilization of these weak interfaces. We conclude by identifying potential target sites at the junction of a weak protein-protein interface.

Cardiolipin is a unique lipid molecule, which has an unusual dimeric structure with a dual-phosphate headgroup and four acyl chains. In most eukaryotic membranes cardiolipin is absent or present only at very low concentrations, however in the inner mitochondrial membrane (IMM) cardiolipin constitutes approximately 10-20% the lipid composition. Cardiolipin is believed to play a role the organization and morphology of the IMM, which contains highly curved membrane regions called cristae that protrude into the matrix. Cardiolipin is also involved in cellular respiration and apoptosis, and has been shown to bind preferentially to several mitochondrial integral and peripheral proteins. During its biosynthesis in the IMM, cardiolipin undergoes a remodeling process whereby acyl chains are removed and replaced to produce mature cardiolipin species with highly unsaturated and uniform lipid tail composition. In the mitochondrial disorder Barth syndrome, defects in the remodeling machinery lead to accumulation of the three-tailed cardiolipin variant monolysocardiolipin. The phenotype of Barth syndrome cells includes abnormal mitochondrial morphology and inhibited energy metabolism. In this work we explore the relationship between the spatial distribution of lipid species and membrane curvature in cardiolipin containing heterogeneous membranes using molecular dynamics simulations. We investigate cardiolipin-containing bilayer properties both in flat lamellar environments and in highly curved (buckled) membranes, and demonstrate that clustering of cardiolipin occurs in areas of high negative curvature. We compare this clustering behavior as well as the pressure induced buckling susceptibility and other biophysical membrane properties in several bilayer systems. One goal of this study is to elucidate the effect of an accumulation of monolysocardiolipin on the physical properties of lipid bilayers.

Szeto-Schiller (SS) peptides are synthetic tetrapeptides containing an alternating aromatic-cationic motif. They are readily taken up by cells and are targeted to the mitochondrial inner membrane. Among them, SS-31 has been shown to bind to bilayers in a cardiolipin-dependent manner via electrostatic and hydrophobic interactions. As a therapeutic agent, SS-31 leads to clinical improvements in many complex diseases, likely linked to its selective interaction with the cardiolipin-rich inner membrane, protection of mitochondrial ultrastructure, curtailment of ROS, and restoration of oxidative phosphorylation. SS-31 represents a first-in-class cardiolipin-protective therapeutic; however, its molecular mechanism of action remains largely unknown. In this study we have explored the use of SS-31 as a potential therapeutic for Barth syndrome (BTHS), a multisystem disorder characterized by cardiac and skeletal myopathy, neutropenia, growth retardation, and premature death in young males. BTHS is caused by mutations in the gene that encodes the transacylase tafazzin, involved in acyl chain remodeling of cardiolipin following its biosynthesis. Dysfunctional tafazzin results in decreased levels of cardiolipin with abnormal fatty acid tail profiles, and an accumulation of the remodeling intermediate monolysocardiolipin. Using model membrane systems and in organello studies, we show that the binding affinities of SS-31 to bilayers containing cardiolipin and monolysocardiolipin are similar. Moreover, the presence of SS-31 appears to modulate the interaction of the electron carrier cytochrome c with bilayers containing cardiolipin and its variants. Our results suggest that SS-31 may act in part by altering membrane phase behavior. Our empirical results are complemented by computational approaches that address the nature of the SS-31 bilayer interaction. Our results point to the potential use of SS peptides as therapeutics for BTHS and give key mechanistic insights into the nature of their bilayer interactions.

A new instrument for production and analysis of samples created by mass-selected ion beam deposition is described. The deposition beamline makes use of a novel phase-space compressor that improves the focusability of the beam at low energies, allowing use with low current cluster ion sources. To illustrate the method, our initial results are presented for deposition of Cu+ and Cu2+ on molybdenum over the energy range from 6 to 220 eV. It is found that deposition of Cu+ at low impact energies results in Cu depositing on the surface with sticking probability of ∼0.6. As the energy is increased above ∼100 eV, an increasing fraction of subsurface copper is observed. In the Cu/Mo system, diffusion of subplanted Cu to the surface is thermodynamically favored, and this probably accounts for the observation of no subplanted Cu at impact energies up to 100 eV. For Cu2+, the results are quite different. The apparent deposition efficiency is similar to that observed for Cu+, however, a significant fraction of sub-surface copper is observed at energies down to ∼40 eV (20 eV/atom). This greater tendency for production of subplanted copper is tentatively rationalized as resulting from a greater propensity for dimer impact to produce complex defects that stabilize subplanted copper. Possible production mechanisms for such defects are discussed.

We have developed an ion beamline for hyperthermal ion-surface collisions that incorporates a phase-space compressor to improve the focusability/current density of the ion beam in the 1–10 eV deposition energy range. In essence, collisional damping is used to substantially improve the brightness of the ion source. In addition to the focusing behavior, the beamline also accomplishes mass selection, source-target pressure reduction of >1010, confinement of the beam to avoid space charge spreading, and hyperthermal energy beam transport. For our application the requirement is moderately tight (100 s of microns) focusing at hyperthermal energies (1–100 eV), but the principle should also be applicable to improving spot sizes/current densities at higher energies.

Dehydrated zeolites NaA, NaY, and barium-exchanged NaY luminesce when irradiated with the 1064 nm laser light of an FT-Raman instrument. When hydrogen is adsorbed in the zeolite, the luminescence is altered in several ways. Most remarkable is the appearance of "absorptions" in the positions of the first vibrational overtones of H 2 and HD. Although these features are in the expected positions with reasonable band profiles for overtone absorptions, the large extinction of the luminescent intensity requires a more efficient mechanism than simple reabsorption of emitted photons. In addition to the appearance of holes in the luminescence spectrum, other luminescent features are substantially quenched by the presence of hydrogen or, in one case, augmented.

The vibrational spectrum of a computer model of crystalline RDX was studied using a 216-molecule periodic supercell, allowing for intra- and intermolecular degrees of freedom using the force field by Boyd et al. [J. Chem. Phys. 124, 104508 (2006)]. The normal modes were analyzed with regard to their activity involving molecule center-of-mass translations and rotations, as well as 15 intramolecular degrees of freedom, including bond stretches, bend and dihedral angle variations, and out-of-plane motions of the nitro groups. We correlate center-of-mass motions with the occupation of internal degrees of freedom for all of the normal modes in the model with particular attention to correlations between nitro rotations and lattice modes. Transfer of lattice energy to internal degrees of freedom can occur through doorway modes and is significant for the initiation of detonation. Several clusters of potential doorway modes are found which involve significant lattice motion as well as nitro rotations. Such groups of modes have been found in the ranges between 186 and 220 and between 420 and 434 cm(-1). Symmetry properties and details of the involved molecular motions are described.

A design is reported for a system of transferrable sample holders and mating experimental stations, that allows samples to be transferred between preparation and analysis tools with high positional accuracy. The sample holders are designed to be manipulated/transported using standard “ESCA stub” compatible hardware. Provisions are made for heating, cooling, and temperature measurement.