Proteins In Biological Membranes Research Articles

Ultrahigh Lithium Selective Transport in Two-Dimensional Confined Ice.

Inspired by selective ion transport in biological membrane proteins, researchers developed artificial ion channels that sieve monovalent cations, catering to the increasing lithium demand. In this work, we engineered an ion transport channel based on the confined ice within two-dimensional (2D) capillaries and found that the permselectivity of monovalent cations depends on the anisotropy of the confined ice. Particularly, the 2D confined ice showed an anomalous lithium selective transport along the (002) direction in the vermiculite capillary, with the Li+/Na+ and Li+/K+ permselectivity reaching up to 556 ± 86 and 901 ± 172, respectively, superior to most ion-selective channels. However, the 2D confined ice along the (100) direction showed less Li+ permselectivity. Additionally, the anisotropy of 2D confined ice can be tuned by adjusting the interlayer spacing. By providing insights into the ion transport in the 2D confined ice, our work may inspire more design of monovalent ion-selective channels for efficient lithium separation.

Triggered Assembly of a DNA-Based Membrane Channel.

Chemistry is in a powerful position to synthetically replicate biomolecular structures. Adding functional complexity is key to increase the biomimetics’ value for science and technology yet is difficult to achieve with poorly controlled building materials. Here, we use defined DNA blocks to rationally design a triggerable synthetic nanopore that integrates multiple functions of biological membrane proteins. Soluble triggers bind via molecular recognition to the nanopore components changing their structure and membrane position, which controls the assembly into a defined channel for efficient transmembrane cargo transport. Using ensemble, single-molecule, and simulation analysis, our activatable pore provides insight into the kinetics and structural dynamics of DNA assembly at the membrane interface. The triggered channel advances functional DNA nanotechnology and synthetic biology and will guide the design of controlled nanodevices for sensing, cell biological research, and drug delivery.

Polyunsaturated Fatty Acids Mediated Regulation of Membrane Biochemistry and Tumor Cell Membrane Integrity.

Particular dramatic macromolecule proteins are responsible for various cellular events in our body system. Lipids have recently recognized a lot more attention of scientists for understanding the relationship between lipid and cellular function and human health However, a biological membrane is formed with a lipid bilayer, which is called a P–L–P design. Our body system is balanced through various communicative signaling pathways derived from biological membrane proteins and lipids. In the case of any fatal disease such as cancer, the biological membrane compositions are altered. To repair the biological membrane composition and prevent cancer, dietary fatty acids, such as omega-3 polyunsaturated fatty acids, are essential in human health but are not directly synthesized in our body system. In this review, we will discuss the alteration of the biological membrane composition in breast cancer. We will highlight the role of dietary fatty acids in altering cellular composition in the P–L–P bilayer. We will also address the importance of omega-3 polyunsaturated fatty acids to regulate the membrane fluidity of cancer cells.

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

The effect of hydrophobic alkyl sidechains on size and solution behaviors of nanodiscs formed by alternating styrene maleamic copolymer

The development of amphipathic polymers, including various formulations of styrene-maleic acid (SMA) copolymers, has allowed the purification of increasing sizes and complexities of biological membrane protein assemblies in native nanodiscs. However, the factors determining the sizes and shapes of the resulting bio-nano particles remain unclear. Here, we show how grafting on short alkyl amine sidechains onto the polar residues leads to a broad set of nanoparticle sizes with improved solution behavior. The solubilization of lipid vesicles occurs over a wide range of pH levels and calcium concentrations, providing utility across the physiologically relevant range of solution conditions.Furthermore, the active SMA derivatives can contain strictly alternating monomers, which have inherently lower sequence polydispersity. Pronounced differences in the shapes of native nanoparticles were formed from Escherichia coli bacterial outer membrane containing PagP protein using methyl, ethyl and propylamine derivatives of styrene-maleic anhydride. In particular, the methylamine-substituted polymer forms smaller, monodispersed nanodiscs, while the longer alkyl derivatives form worm-like nanostructures. Thus the introduction of hydrophobicity onto the polar sidechains of amphipathic polymers has profound effects on morphology of native nanodisc, with shorter methyl moieties offering more uniformity and utility for structural biology studies.

Editorial: Biographical Memoirs , Volume 68

Editorial: <i>Biographical Memoirs</i> , Volume 68

David Christopher Gadsby. 26 March 1947—9 March 2019

Over nearly five decades, David Christopher Gadsby pioneered biophysical research that advanced our mechanistic understanding of ion-transporting proteins in biological membranes. His passion for hands-on do-it-yourself electrophysiology, his depth of analytical rigor, and his idiosyncratic scientific aesthetic expanded the edge of discovery in two areas: the electrical character of the Na + pump, and the molecular workings of ‘cystic fibrosis transmembrane regulator’ (CFTR), the chloride ion channel whose mutations cause cystic fibrosis. His approach was flavoured by an appreciation for common underlying features between these ostensibly distinct types of membrane-transport systems. While David's focus was first on the basic molecular biophysics of a problem, he was always attuned to implications of his discoveries for human health. Based in New York at The Rockefeller University throughout his independent scientific career, and at the Marine Biological Laboratory in Woods Hole, Massachusetts, as a squid-season research-scientist, he was proficient in wrestling with problems spanning a wide swath of membrane biology: from determinants of the cardiac electrical waveform, to microsecond-timescale ionic currents in squid axons, to details of structure–mechanism relations in membrane pump and ion-channel proteins. He wore his eminence lightly and never distanced himself from the laboratory, where he often performed experiments with his own hands right up to his retirement. His reserved scientific personality, which demanded equally from his colleagues and himself immaculate data, unclouded logic, and substantive pertinence to the issues at hand, contrasted with his palpable joy in a good experiment and in his sea-loving life outside the lab.

Transmembrane Peptide Insertion Affects Membrane Interfacial Dynamics

Macromolecular crowding in biological context has received significant attention recently, as it has become clear that many of our experimental approaches using dilute aqueous solutions of biomolecules are quite simplistic compared to true biological systems. True biological environments are tightly packed, with biomolecular concentrations ranging upwards of 400 mg/mL, and this crowding has been shown to have a profound effect on processes such as protein folding, stability, and small molecule binding. While tremendous focus has been placed on crowding effects on cytoplasm and proteins themselves, it follows that biological membranes are also susceptible to structural, functional, and dynamical consequences of crowding, and there is no question that the large volume of proteins in biological membranes contributes to this. Here, we explore these consequences in model lipid bilayers, probing lipid hydration, hydrogen bond networks, and hydrogen bond lifetimes at the lipid-water interface and their dependence on transmembrane peptide concentration. To do so, we have employed two-dimensional infrared spectroscopy (2D IR) in conjunction with molecular dynamics simulations to directly probe the lipid-water interface of DMPC vesicles with various loading concentrations of pH (Low) Insertion Peptide (pHLIP). We have found experimentally non-monotonic dependence of interfacial dynamics as a function of increasing pHLIP concentration: at low peptide concentrations dynamics are faster than in pure DMPC, whereas at high - approaching biologically relevant - concentrations, dynamics are significantly slowed. Using our computational models, we determine that this phenomenon is rooted in water ordering at the interface. In pure DMPC vesicles, simulations reveal ice-like water ordering at the interface. At low peptide concentrations, interfacial water becomes disordered leading to fast dynamics, and at high peptide concentration the water molecules are orientationally constrained, effectively dehydrating the interface.

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

The expression and integration of membrane proteins into vesicle membranes is a critical step in the design of cell-mimetic biosensors, bioreactors, and artificial cells. While membrane proteins have been integrated into a variety of nonnatural membranes, the effects of the chemical and physical properties of these vesicle membranes on protein behavior remain largely unknown. Nonnatural amphiphiles, such as diblock copolymers, provide an interface that can be synthetically controlled to better investigate this relationship. Here, we focus on the initial step in a membrane protein's life cycle: expression and folding. We observe improvements in both the folding and overall production of a model mechanosensitive channel protein, the mechanosensitive channel of large conductance, during cell-free reactions when vesicles containing diblock copolymers are present. By systematically tuning the membrane composition of vesicles through incorporation of a poly(ethylene oxide)-b-poly(butadiene) diblock copolymer, we show that membrane protein folding and production can be improved over that observed in traditional lipid vesicles. We then reproduce this effect with an alternate membrane-elasticizing molecule, C12E8 Our results suggest that global membrane physical properties, specifically available membrane surface area and the membrane area expansion modulus, significantly influence the folding and yield of a membrane protein. Furthermore, our results set the stage for explorations into how nonnatural membrane amphiphiles can be used to both study and enhance the production of biological membrane proteins.

NANODISCS: A NEW EPOCH IN THE STUDY OF MEMBRANE PROTEINS AND AS AN EMERGING DRUG DELIVERY SYSTEM

Nano discs recently evolved as a novel tool for studying the membrane associated proteins and serve as an effective drug delivery system. Nano discs constitute disc shaped nano particles and can be defined as a membrane system which is synthetic in nature and aids in the study of membrane proteins. It is mainly made of phospholipid bilayer and the water repelling edge is isolated by amphipathic proteins called membrane scaffolding proteins [MSP]. Micelles present in the nano disc mimics the property of the biological membrane proteins. It is a powerful technology that competently delivers the drug components in to the right cells in the right tissues. Membrane scaffold proteins are primarily expressed, purified and characterized and self-assembled to form Nano discs by the process of dialysis using biobeads. Nano discs are proven to be effective in the study of membrane proteins because they can fluidize and counterbalance and also help in reclusion, refinement, biophysical and biochemical studies of them. It also presents a more genuine environment than liposomes, bicelles, amphipols and detergent micelles. Major technological advantages of nano discs include the higher stability and carrier capacity and also the increased feasibility of incorporating both hydrophilic and hydrophobic substances of drug carrier. Thus nano discs serves as an excellent system in its ability to precisely control its composition and provide a nano scale membrane surface for investigating molecular recognition events. This article reviews the emphasis of nanodiscs in studying membrane proteins as well as its effectivity in transforming into a major drug delivery system. An overview of published literatures between 1996 and 2017 was conducted to write the review.

Transmembrane Electric Conductivity Modulation in PCBM Doped Free-Standing Lipid Bilayers By Visible Light Irradiation

A cell membrane has a thickness of several nanometer and function as an insulating film having a resistance of more than 100 GΩ in a biological body. The cell membrane has a structure in which phospholipid molecules are aligned as a bilayer. Researches have been conducted to artificially construct cell membranes in vivo and to apply them as highly sensitive biosensors and drug screening devices by embedding biological membrane proteins in cell membranes. On the other hand, photovoltaic properties of lipid bilayer membranes have been reported by introducing fullerene (C60) in the lipid bilayer membrane. Although this technique suggests the function of the cell membrane as a photosensor, the mechanism has not been discussed yet. If we modulated the trans-electrical-conduction with external energies of electric filed or light irradiation, we could expect to control of carrier and/or ion incorporation at the cell membrane, that would be used a biosensing and bio-ion-incorporation devices. In this work, we investigated that modulation of transmembrane electric conductivity in Phenyl-C61-Butyric-Acid-Methyl Ester (PC60BM) doped free-standing lipid bilayers by the visible light irradiation.

Curvature Mediated Interactions in Highly Curved Membranes

Cellular membranes exhibit a variety of morphologies, from a simple spherical liposome to bewildering complex structures like interconnected tubular networks found in the Endoplasmic Reticulum, or connected stacks of perforated membrane sheets as in the Golgi apparatus. Membranes form these structures through different mechanisms, the most important of which is through the interplay between membrane lipids and inclusions like proteins. Spatial organization of such proteins in biological membranes is essential for stabilizing the membrane and for the dynamic behavior of cellular organelles. Despite making significant strides in understanding membrane mediated interactions, it is not completely understood how inclusions interact in a highly curved regime. It is, therefore, warranted to investigate how proteins in strongly bent membranes self-organize. We do so using both analytical calculations based on the course-grained Helfrich model, and with Monte Carlo simulations. First, we find that the forces between identical inclusions, which are known to be always repulsive on a flat membrane, become attractive on curved and closed membranes. Second, we show how many point-like inclusions embedded in a tubular membrane spontaneously self-assemble into rings and lines. Finally, we quantitatively show how inclusions adhered to an ellipsoidal membrane likewise self-assemble into a ring perpendicular to its semi-major axis. The structures we find are highly relevant in biology, and are found for instance in endocytosis and cell division. Our results therefore reveal that purely physical interactions underlie these biological phenomena.

Saturation-Recovery EPR Spin-Labeling Method for Quantification of Lipids in Domains of Biological Membranes

The presence of integral membrane proteins in biological membranes induces the formation of distinct domains in the lipid bilayer portion of these membranes. We were able to discriminate the existence of three lipid environments in intact fiber cell plasma membranes of the eye lens, namely bulk, boundary, and trapped lipids. However, our qualitative approach did not allow quantification of lipids in each domain. Recently, based on the continuous wave (CW) electron paramagnetic resonance (EPR) spectra of phospholipid analog and cholesterol analog spin labels, we developed a method that allows evaluation of the relative amount of phospholipids and cholesterol in the bulk lipid domain and in the boundary plus trapped lipid domain [M. Raguz, L. Mainali,W. J. O’Brien and W. K. Subczynski, (2015), Exp. Eye Res., 140:179-186]. Here, we will present a new approach that, based on saturation-recovery (SR) EPR, allows evaluation of the relative amount of phospholipids and cholesterol in the bulk plus boundary lipid domain and in the trapped lipid domain. Spectrometer conditions for the successful application of this method to quantify lipids in membrane domains are described. These two methods clearly demonstrate that the method's time window allows the separation of results from the different domains. CW EPR mixes results from the boundary and trapped lipids, while SR EPR mixes results from the bulk and boundary lipids. These two methods complement each other, providing a more complete picture of lipid lateral organization in intact membranes. The abilities of these methods are illustrated in the intact fiber cell plasma membranes from porcine eye lenses.Acknowledgments: This work was supported by grants EY015526, EB001980, and EY001931 from the National Institutes of Health.

Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

Suppressing membrane height fluctuations leads to a membrane-mediated interaction among proteins.

Membrane-induced interactions can play a significant role in the spatial distribution of membrane-bound proteins. We develop a model that combines a continuum description of lipid bilayers with a discrete particle model of proteins to probe the emerging structure of the combined membrane-protein system. Our model takes into account the membrane's elastic behavior, the steric repulsion between proteins, and the quenching of membrane shape fluctuations due to the presence of the proteins. We employ coupled Langevin equations to describe the dynamics of the system. We show that coupling to the membrane induces an attractive interaction among proteins, which may contribute to the clustering of proteins in biological membranes. We investigate the lateral protein diffusion and find that it is reduced due to transient fluctuations in membrane shape.

Hydrodynamic collective effects of active proteins in biological membranes.

Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015)PNASA60027-842410.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them.

In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.

Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution.

Revisiting the curvature-mediated interactions between proteins in biological membranes.

Proteins embedded in soft biological membranes experience a long-range force mediated by elastic curvature deformations. The classical linearized Helfrich-Canham Hamiltonian based derivations reveal the nature of the force between a pair of proteins to be repulsive in the zero-temperature limit and the interaction potential is inversely proportional to the fourth power of the distance separating the inclusions. Such a result is the starting point to understand many-body interactions between proteins in biological membranes and the study of their clustering or, more broadly, self-organization. A key observation regarding this widely quoted result is that any two (mechanically rigid) proteins will experience an identical force. In other words, there is no specificity in the currently employed continuum models that purport to explain protein interactions. In this work we argue that each protein has a unique mechanical signature based on its interaction with the surrounding lipid bilayer membrane and cannot be treated as a non-specific rigid object. We modify the classical Helfrich-Canham theory of curvature elasticity to incorporate protein-membrane specificity, discuss the estimation of the new model parameters via atomistic simulations and re-evaluate the curvature-mediated force between proteins. We find that the incorporation of protein-specificity can reduce the interaction force by several orders of magnitude. Our result may provide at least one plausible reason behind why in some computational and experimental studies, a net attractive force between proteins is in evidence.

Observing Peptide-Induced Lipid Accumulation in a Single-Component Zwitterionic Lipid Bilayer

Membrane domain formation plays a key role in various cellular functions and biological events. Lateral accumulation of lipids and proteins in biological membranes is one of the most important factors that control the domain formation. However, compared to numerous reports on the lipid aggregation or accumulation formed in the membranes composed of multiple components of lipids and cholesterol, the lipid accumulation in one-component phospholipid bilayer system is still rare. In this study, we demonstrate that short peptides can induce the lipid accumulation in a single-component zwitterionic lipid bilayer. By investigating the interaction between a short peptide of mastoparan (MP, a G-protein-activating peptide) and neutral phosphocholine lipid bilayers using sum frequency generation vibrational spectroscopy (SFG-VS), we have found that MP can cause a local accumulation of lipid molecules at the outer leaflet of the lipid bilayer, resulting in more than 10 times intensity increase in the signals from the...

Dynamics of Membrane Proteins within Synthetic Polymer Membranes with Large Hydrophobic Mismatch.

The functioning of biological membrane proteins (MPs) within synthetic block copolymer membranes is an intriguing phenomenon that is believed to offer great potential for applications in life and medical sciences and engineering. The question why biological MPs are able to function in this completely artificial environment is still unresolved by any experimental data. Here, we have analyzed the lateral diffusion properties of different sized MPs within poly(dimethylsiloxane) (PDMS)-containing amphiphilic block copolymer membranes of membrane thicknesses between 9 and 13 nm, which results in a hydrophobic mismatch between the membrane thickness and the size of the proteins of 3.3-7.1 nm (3.5-5 times). We show that the high flexibility of PDMS, which provides membrane fluidities similar to phospholipid bilayers, is the key-factor for MP incorporation.