Modulation of lipid domain formation in mixed model systems by proteins and peptides

The control of lipid domain formation in biological membranes has received limited consideration. This mechanism is quantitatively investigated using Monte Carlo computer simulations of a simple model system. Monte Carlo simulations are performed on a simple model system composed of phosphatidylecholine (PC), phosphatidylserine (PS), and cholesterol (Chol). Domain formation induced by binding of the phospholipid binding proteins, annexin A5 (A5) and the C2 protein motif is investigated. Simulations for models containing PC/PS lipids indicate that the addition of A5 does not induce lipid domain formation while binding of C2 greatly induces lipid domain formation. The addition of Chol to PC/PS systems was found to induce lipid demixing in the absence and presence of A5 and further enhance the ability of C2 to form PS domains. Incorporation of a preferential protein-protein interaction to PC/PS and PC/PS/Chol systems was found to further increase lipid demixing for all compositions. Lipid domain formation is also investigated experimentally using fluorescence resonance energy transfer (FRET) in 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ sphingomyelin from porcine brain (BSM)/ Cholesterol (Chol) model systems. Studies have shown that these model systems contain lipid domains. The dependence of lipid domain size upon the addition of the transmembrane region of the linker for activation of T-cells (LAT), a protein believed to associate with lipid rafts, is investigated. When incorporated, LAT was found to insert into both the liquid-ordered (Lo) and liquid-disordered (Ld) regions indicating no lipid specificity. FRET between an acceptor/donor pair shown to not be affected by addition of LAT in POPC/BSM/Chol mixtures indicating that the presence of LAT does not affect the size of lipid domains

Magnetism, FeS colloids, and Origins of Life

A number of features of living systems: reversible interactions and weak bonds underlying motor-dynamics; gel-sol transitions; cellular connected fractal organization; asymmetry in interactions and organization; quantum coherent phenomena; to name some, can have a natural accounting via $physical$ interactions, which we therefore seek to incorporate by expanding the horizons of `chemistry-only' approaches to the origins of life. It is suggested that the magnetic 'face' of the minerals from the inorganic world, recognized to have played a pivotal role in initiating Life, may throw light on some of these issues. A magnetic environment in the form of rocks in the Hadean Ocean could have enabled the accretion and therefore an ordered confinement of super-paramagnetic colloids within a structured phase. A moderate H-field can help magnetic nano-particles to not only overcome thermal fluctuations but also harness them. Such controlled dynamics brings in the possibility of accessing quantum effects, which together with frustrations in magnetic ordering and hysteresis (a natural mechanism for a primitive memory) could throw light on the birth of biological information which, as Abel argues, requires a combination of order and complexity. This scenario gains strength from observations of scale-free framboidal forms of the greigite mineral, with a magnetic basis of assembly. And greigite's metabolic potential plays a key role in the mound scenario of Russell and coworkers-an expansion of which is suggested for including magnetism.Comment: 42 pages, 5 figures, to be published in A.R. Memorial volume, Ed Krishnaswami Alladi, Springer 201

Reduced level of docosahexaenoic acid shifts GPCR neuroreceptors to less ordered membrane regions

G protein-coupled receptors (GPCRs) control cellular signaling and responses. Many of these GPCRs are modulated by cholesterol and polyunsaturated fatty acids (PUFAs) which have been shown to co-exist with saturated lipids in ordered membrane domains. However, the lipid compositions of such domains extracted from the brain cortex tissue of individuals suffering from GPCR-associated neurological disorders show drastically lowered levels of PUFAs. Here, using free energy techniques and multiscale simulations of numerous membrane proteins, we show that the presence of the PUFA DHA helps helical multi-pass proteins such as GPCRs partition into ordered membrane domains. The mechanism is based on hybrid lipids, whose PUFA chains coat the rough protein surface, while the saturated chains face the raft environment, thus minimizing perturbations therein. Our findings suggest that the reduction of GPCR partitioning to their native ordered environments due to PUFA depletion might affect the function of these receptors in numerous neurodegenerative diseases, where the membrane PUFA levels in the brain are decreased. We hope that this work inspires experimental studies on the connection between membrane PUFA levels and GPCR signaling. Author summary Our current picture of cellular membranes depicts them as laterally heterogeneous sheets of lipids crowded with membrane proteins. These proteins often require a specific lipid environment to efficiently perform their functions. Certain neuroreceptor proteins are regulated by membrane cholesterol that is considered to be enriched in ordered membrane domains. In the brain, these very same domains also contain a fair amount of polyunsaturated fatty acids (PUFAs) that have also been discovered to interact favorably with many receptor proteins. However, certain neurological diseasesassociated with the inadequate functioning of the neuroreceptorsseem to result in the decrease of brain PUFA levels. We hypothesized that this decrease in PUFA levels somehow inhibits receptor partitioning to cholesterol-rich domains, which could further compromise their function. We verified our hypothesis by an extensive set of computer simulations. They demonstrated that the PUFA-receptor interaction indeed leads to favorable partitioning of the receptors in the cholesterol-rich ordered domains. Moreover, the underlying mechanism based on the shielding of the rough protein surface by the PUFAs seems to be exclusive for multi-helical protein structures, of which neuroreceptors are a prime example.Peer reviewe

Computational Algorithms for Predicting Membrane Protein Assembly From Angstrom to Micron Scale

Biological barriers in the human body are one of the most crucial interfaces perfected through evolution for diverse and unique functions. Of the wide range of barriers, the paracellular protein interfaces of epithelial and endothelial cells called tight junctions with high molecular specificities are vital for homeostasis and to maintain proper health. While the breakdown of these barriers is associated with serious pathological consequences, their intact presence also poses a challenge to effective delivery of therapeutic drugs. Complimenting a rigorous combination of in vitro and in vivo approaches to establishing the fundamental biological construct, in addition to elucidating pathological implications and pharmaceutical interests, a systematic in silico approach is undertaken in this work in order to complete the molecular puzzle of the tight junctions. This work presents a bottom-up approach involving a careful consideration of protein interactions with Angstrom-level details integrated systematically, based on the principles of statistical thermodynamics and probabilities and designed using well-structured computational algorithms, up to micron-level molecular architecture of tight junctions, forming a robust prediction with molecular details packed for up to four orders of magnitude in length scale. This work is intended to bridge the gap between the computational nano-scale studies and the experimental micron-scale observations and provide a molecular explanation for cellular behaviors in the maintenance, and the adverse consequences of breakdown of these barriers. Furthermore, a comprehensive understanding of tight junctions shall enable development of safe strategies for enhanced delivery of therapeutics

Simulating protein evolution via thermodynamic models

Natural proteins are results of evolution and they need to maintain certain thermodynamic stabilities in order to carry out their biological functions. By simulating protein evolution based on thermodynamic rules, we could reconstruct the evolution trajectory and analyze the evolutionary dynamics of a protein population, and further understand the protein sequence-structure-function relationship. In this study, we have used both a simplified lattice model and a high-resolution atomic model to simulate protein evolution processes. With the lattice model, we have investigated general theoretical questions about how protein structural designability would affect protein evolution, particularly how it would affect protein recombination and protein-ligand interactions in the evolution process. With the atomic model, we could simulate evolution processes for particular protein with different selection pressure. First, we simulated directed evolution processes and utilized such model to investigate the thermostabilization of T4 lysozyme. Second, we simulated neutral evolution processes for HIV protease, investigated its evolutionary dynamics and the possible drug-resistance mechanism in such neutral evolution. Overall, thermodynamic models can help us understand either general protein evolution dynamics or specific protein sequence-structure-function relationship in evolution

Extracellular annexin A5: Functions of phosphatidylserine-binding and two-dimensional crystallization

AbstractIn normal healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. However, on activated platelets, dying cells and under specific circumstances also on various types of viable leukocytes phosphatidylserine is actively externalized to the outer leaflet of the plasma membrane. Annexin A5 has the ability to bind in a calcium-dependent manner to phosphatidylserine and to form a membrane-bound two-dimensional crystal lattice. Based on these abilities various functions for extracellular annexin A5 on the phosphatidylserine-expressing plasma membrane have been proposed. In this review we describe possible mechanisms for externalization of annexin A5 and various processes in which extracellular annexin A5 may play a role such as blood coagulation, apoptosis, phagocytosis and formation of plasma membrane-derived microparticles. We further highlight the recent discovery of internalization of extracellular annexin A5 by phosphatidylserine-expressing cells

Structure and dynamics of membrane peptides from solid-state NMR

Solid-state NMR is among the most important analytical techniques to provide atomic-level structural and dynamic information of chemical and biological systems. Due to the insoluble and non-crystalline nature of most membrane peptides and proteins, SSNMR is particularly powerful to investigate their conformations, dynamics, domain assembly, oliogmerization, and the characteristic structural properties in lipid bilayers including insertion orientation and depth, residue-lipid interaction, and membrane per-turbation. Our research is to collect structural and dynamic information and correlate it with biological functions to elucidate the structure-bioactivity relation. In my PhD pro-jects, we have successfully applied various SSNMR techniques to study many interesting membrane peptides including the cell-penetrating peptide (CPP), antimicrobial peptides (AMP), antimicrobial oligomer (AMO), gating helix of K+ channel (KvAP) and trans-membrane 1H channel of influenza M2 protein (M2TM). We also developed a novel paramagnetic-ion-membrane bound paramagnetic relaxation enhancement (PRE) method to provide quantitative long-range distance constrain (~20 y) in membrane-active bio-systems and applied the method to obtain high-resolution residue-specific insertion depth of two membrane peptides, penetratin and M2TM. One main category of my research topics is the cationic membrane peptide. On the one hand, phospholipid membranes have highly hydrophobic interiors that cannot accommodate charged species, while on the other hand, cationic peptides need to insert or translocate across the membrane to conduct biological functions. So, we are motivated to uncover the structural basis of the membrane insertion and translocation. With this motivation, we have studied two kinds of cationic bio-macromolecules, including CPP and AMP. We have experimentally proved that all these Arg-rich peptides generally have strong guanidinium-phosphate interaction with the phospholipids. This charge-charge interaction causes headgroup reorientation and allows the peptide to insert. For CPPs, the guanidinium-phosphate ion pair helps to stabilize the unstructured peptide in the membrane-water interface. The observed peptide-water interaction further minimizes the peptide polarity and makes it more membrane-soluble. We find that two representative CPPs, penetratin and TAT, have highly dynamic and plastic conformations, proposed to facilitate the movement within the membrane. In the penetratin study, the one-side Mn2+-bound PRE method has been developed and applied to study the pep-tide-concentration dependent insertion depth and symmetry in the outer and inner leaflets of the POPC/POPG bilayer. Another important kind of cationic membrane peptides is AMP. Taking PG-1 and its charge reduced mutant IB484 as model AMPs, we have stud-ied the antimicrobial mechanism, and for the first time, provided high-resolution struc-tural information to elucidate the bacterial Gram-selectivity. We find that the interaction manifests the manner of peptide insertion in terms of orientation and depth, which in turn determined the antimicrobial ability in gram positive and negative bacterial membranes. The antimicrobial mechanism of a guanidinium-rich AMO, PMX30016, has also been investigated. The finding of drug-concentration dependant lipid 31P CSA change and the fast uniaxial motion in the interfacial membrane region suggest a subtle and combined antimcicrobial mechanism of membrane potential perturbation and in-plane disruption. Another category of my research topics is the transmembrane ion-conductive channel study, including the gating mechanism of a K+ channel (KvAP) and the blocking mechanism of the M2TM 1H channel by the metal ion inhibitor (Cu2+). We have deter-mined the topology of an isolated gating helix (S4) of KvAP and compared the orientation with that of an intact K+ channel, Kv1.2-Kv2.1 paddle chimera. The identical tilted and rotational angles of the S4 helix in the isolated form and intact protein, and the observed interaction suggest the channel gating might be manifested by the pep-tide-lipid interaction rather than the interaction among different helical domains. Finally, we applied PRE techniques to study the Cu2+-inhibited M2TM channel and obtained high resolution Cu2+ binding structure and long-range distance constraints for the binding structure refinement

Snake Cytotoxins Bind to Membranes via Interactions with Phosphatidylserine Head Groups of Lipids

The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of cells