Permeation of nanocrystals across lipid membranes

Biological membranes are one of the major structural elements of cells, and play a key role as a selective barrier and substrate for many proteins that facilitate transport and signaling processes. Understanding the structural and mechanical properties of lipid membranes during permeation of nanomaterials is of prime importance in determining the toxicity of nanomaterials to living cells. It has been shown that the interaction between lipid membranes and nanomaterials and the disruption of lipid membranes are often determined by physicochemical properties of nanomaterials, such as size, shape and surface composition. In this work, molecular dynamic simulations were carried out using various sizes of nanocrystals as a probe to explore the transport of nanomaterials across dipalmitoylphosphatidylcholine (DPPC) bilayers and the changes in the structural and mechanical properties of DPPC bilayers during the permeation. A coarse-grained model was used to provide insight at large time and length scales. In this work, an external driving force helps the nanocrystals across the lipid bilayer. The minimum forces needed to penetrate the model membrane and the interaction of nanocrystals and lipid bilayers were investigated in simulations. The elastic and dynamic properties of lipid bilayers, including the local and bulk properties during the permeation of the nanocrystals, which are of considerable fundamental interest, were also studied. The findings described will lead to better understanding of nanomaterial–lipid membrane interactions and the mechanical and dynamic properties of lipid membranes under permeation.     

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

The preparation of oriented, hydration-optimized lipid bilayer samples, for NMR structure determination of membrane proteins, is described. The samples consist of planar phospholipid bilayers, containing membrane proteins, that are oriented on single pairs of glass slides, and are placed in the coil of the NMR probe with the bilayer plane perpendicular to the direction of the magnetic field. Lipid bilayers provide a medium that closely resembles the biological membrane, and sample orientation both preserves the intrinsic membrane-defined directional quality of membrane proteins, and provides the mechanism for resonance line narrowing. The hydration-optimized samples overcome some of the difficulties associated with multi-dimensional, high-resolution, solid-state NMR spectroscopy of membrane proteins. These samples have greater stability over the course of multi-dimensional NMR experiments, they have lower sample conductance for greater rf power efficiency, and enable greater rf coil filling factors to be obtained for improved experimental sensitivity. Sample preparation is illustrated for the membrane protein CHIF (channel inducing factor), a member of the FXYD family of ion transport regulators.     

Using low-E resonators to reduce RF heating in biological samples for static solid-state NMR up to 900 MHz

RF heating of solid-state biological samples is known to be a destabilizing factor in high-field NMR experiments that shortens the sample lifetime by continuous dehydration during the high-power cross-polarization and decoupling pulses. In this work, we describe specially designed, large volume, low-E 15N-1H solid-state NMR probes developed for 600 and 900 MHz PISEMA studies of dilute membrane proteins oriented in hydrated and dielectrically lossy lipid bilayers. The probes use an orthogonal coil design in which separate resonators pursue their own aims at the respective frequencies, resulting in a simplified and more efficient matching network. Sample heating at the 1H frequency is minimized by a loop-gap resonator which produces a homogeneous magnetic field B1 with low electric field E. Within the loop-gap resonator, a multi-turn solenoid closely matching the shape of the sample serves as an efficient observe coil. We compare power dissipation in a typical lossy bilayer sample in the new low-E probe and in a previously reported 15N-1H probe which uses a double-tuned 4-turn solenoid. RF loss in the sample is measured in each probe by observing changes in the 1H 360 degrees pulse lengths. For the same values of 1H B1 field, sample heating in the new probe was found to be smaller by an order of magnitude. Applications of the low-E design to the PISEMA study of membrane proteins in their native hydrated bilayer environment are demonstrated at 600 and 900 MHz.     

Fluorescence techniques for probing water penetration into lipid bilayers

Fluorescence spectroscopy can be used as a highly sensitive and localized probe for hydration in lipid bilayers. Water associates with the head-group region, where it participates in an interlipid network of hydrogen bonds. Deeper in the bilayer, water is contained within acyl-chain packing defects. Fluorescence methodology is available to probe both the interstitial and head-group hydration in lipid bilayers, and results are in good agreement with other techniques. Using fluorescence spectroscopic approaches, cholesterol is shown to dehydrate the acyl-chain region, while hydrating the head-group region. Membrane proteins appear to increase acyl-chain hydration at the protein-lipid interface. Overall fluorescence spectroscopic techniques may be most effective in studying the water content of lipid bilayers and especially of biological membranes.     

Nanomechanical properties of single amyloid fibrils

Amyloid fibrils are traditionally associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease or Creutzfeldt-Jakob disease. However, the ability to form amyloid fibrils appears to be a more generic property of proteins. While disease-related, or pathological, amyloid fibrils are relevant for understanding the pathology and course of the disease, functional amyloids are involved, for example, in the exceptionally strong adhesive properties of natural adhesives. Amyloid fibrils are thus becoming increasingly interesting as versatile nanobiomaterials for applications in biotechnology. In the last decade a number of studies have reported on the intriguing mechanical characteristics of amyloid fibrils. In most of these studies atomic force microscopy (AFM) and atomic force spectroscopy play a central role. AFM techniques make it possible to probe, at nanometer length scales, and with exquisite control over the applied forces, biological samples in different environmental conditions. In this review we describe the different AFM techniques used for probing mechanical properties of single amyloid fibrils on the nanoscale. An overview is given of the existing mechanical studies on amyloid. We discuss the difficulties encountered with respect to the small fibril sizes and polymorphic behavior of amyloid fibrils. In particular, the different conformational packing of monomers within the fibrils leads to a heterogeneity in mechanical properties. We conclude with a brief outlook on how our knowledge of these mechanical properties of the amyloid fibrils can be exploited in the construction of nanomaterials from amyloid fibrils.     

磷脂跨膜交换机制的研究

膜间磷脂交换是一项重要的生理活动, 其对药物运输及膜功能研究有重要意义. 本文用石英晶体微天平及耗散系数测试仪研究囊泡与囊泡、囊泡与支撑膜间磷脂交换行为, 荧光光谱仪用来测量膜表面电性与膜组分对磷脂交换的影响. 实验结果表明: 磷脂跨膜交换速率与交换时间成反比, 膜表面异电性磷脂的增加会加速膜内相互作用和磷脂跨膜交换速率, 以及改变膜表面组分会对囊泡与支撑膜间的磷脂交换产生影响. 本文研究有助于加深理解磷脂跨膜交换机制, 并对药学研究提供参考.     

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.     

Membranes,peptides, and disease: Unraveling the mechanisms of viral proteins with solid state nuclear magnetic resonance spectroscopy

The interplay between peptides and lipid bilayers drives crucial biological processes. For example, a critical step in the replication cycle of enveloped viruses is the fusion of the viral membrane and host cell endosomal membrane, and these fusion events are controlled by viral fusion peptides. Thus such membrane-interacting peptides are of considerable interest as potential pharmacological targets. Deeper insight is needed into the mechanisms by which fusion peptides and other viral peptides modulate their surrounding membrane environment, and also how the particular membrane environment modulates the structure and activity of these peptides. An important step toward understanding these processes is to characterize the structure of viral peptides in environments that are as biologically relevant as possible. Solid state nuclear magnetic resonance (ssNMR) is uniquely well suited to provide atomic level information on the structure and dynamics of both membrane-associated peptides as well as the lipid bilayer itself; further ssNMR can delineate the contribution of specific membrane components, such as cholesterol, or changing cellular conditions, such as a decrease in pH on membrane-associating peptides. This paper highlights recent advances in the study of three types of membrane associated viral peptides by ssNMR to illustrate the more general power of ssNMR in addressing important biological questions involving membrane proteins.     

The New Fluorescent Membrane Probe Ahba: A Comparative Study with the Largely Used Laurdan

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.     

Development of the field of structural physiology

Electron crystallography is especially useful for studying the structure and function of membrane proteins — key molecules with important functions in neural and other cells. Electron crystallography is now an established technique for analyzing the structures of membrane proteins in lipid bilayers that closely simulate their natural biological environment. Utilizing cryo-electron microscopes with helium-cooled specimen stages that were developed through a personal motivation to understand the functions of neural systems from a structural point of view, the structures of membrane proteins can be analyzed at a higher than 3 Å resolution. This review covers four objectives. First, I introduce the new research field of structural physiology. Second, I recount some of the struggles involved in developing cryo-electron microscopes. Third, I review the structural and functional analyses of membrane proteins mainly by electron crystallography using cryo-electron microscopes. Finally, I discuss multifunctional channels named “adhennels” based on structures analyzed using electron and X-ray crystallography.     

Organization of membrane-associated proteins in lipid bilayers

Lateral organization of proteins in biomembranes is vitally important to membrane functions such as signal transduction, endocytosis, and membrane trafficking. One of the major goals in current biomembrane science is to reveal the microscopic mechanism of membrane-associated protein organization in biomembranes. Here, we investigate the structural organization of membrane-associated proteins in lipid bilayers by combining self-consistent field theory with density functional theory. The present study can simultaneously take into account the entropy effect of lipids, depletion effect of membrane-associated proteins due to the presence of lipid headgroups as well as the effect of interfacial interaction. By varying the volume fraction of lipids, we examine various effects on protein organization, and reveal that a close-packed crystal structure appears at low lipid volume fractions due to interfacial energy and weak depletion effect, whereas a chain structure with branches occurs at high lipid volume fractions mainly due to strong depletion. The present results may provide some theoretical insight into further experiments on organization of membrane proteins.     

Ultrasonic studies of membrane suspensions

Summary The mechanical and the dynamic properties of various membranes were studied by the ultrasonic-resonance method. The compressibility of the membranes was in the range from 2.5·10⁻¹¹ to 5·10⁻¹¹ cm²/dyn and influenced by several factors such as phase transition, incorporation of cholesterol or proteins and configuration of proteins. The ultrasonic relaxation was measured for two membrane systems, dipalmitoylphosphatidylcholine bilayer and sarcoplasmic-reticulum membrane. The results indicated that the ultrasonic measurements are very sensitive to the co-operative transition in lipid bilayers as well as the relaxation of biological membranes containing intrinsic proteins. Paper presented at the ?Meeting on Lyotropics and Related Fields?, held in Rende, Cosenza, September 13–18, 1982.     

Bilayers of neutral lipids bear a small but significant charge

Many experiments done on neutral lipid bilayers in pure water show weak repulsions. These weak forces prevent vesicles from adhering and are generally overcome by adding some salt in the aqueous medium. They also appear as stray repulsions in surface forces measurements made on lipid bilayers. Using a surface forces apparatus in pure water and in salt solution, we have measured the forces between two stearoyl-oleoyl-phosphatidyl-choline (SOPC) bilayers and between two dimiristoyl-phosphatidyl-ethanolamine (DMPE) bilayers. The results show that the repulsions are due to a small amount of negative charges coming from impurities in SOPC. This was quantitatively confirmed by electrophoretic measurements. There are 3 times less charges in the case of DMPE layers. The effect of these charges which is negligible at high salt concentration may significantly affect the adhesion energy and behaviour of neutral lipid bilayers between 0 and salt. Received 18 December 1998     

Atomic force microscopy for the investigation of molecular and cellular behavior

The present review details the methods used for the measurement of cells and their exudates using atomic force microscopy (AFM) and outlines the general conclusions drawn by the mechanical characterization of biological materials through this method. AFM is a material characterization technique that can be operated in liquid conditions, allowing its use for the investigation of the mechanical properties of biological materials in their native environments. AFM has been used for the mechanical investigation of proteins, nucleic acids, biofilms, secretions, membrane bilayers, tissues and bacterial or eukaryotic cells; however, comparison between studies is difficult due to variances between tip sizes and morphologies, sample fixation and immobilization strategies, conditions of measurement and the mechanical parameters used for the quantification of biomaterial response. Although standard protocols for the AFM investigation of biological materials are limited and minor differences in measurement conditions may create large discrepancies, the method is nonetheless highly effective for comparatively evaluating the mechanical integrity of biomaterials and can be used for the real-time acquisition of elasticity data following the introduction of a chemical or mechanical stimulus. While it is currently of limited diagnostic value, the technique is also useful for basic research in cancer biology and the characterization of disease progression and wound healing processes.     

On the Membrane Binding of the Potentiometric Probe Di-4-ANEPPS: A Fluorescence and Resonance Raman Study

Di-4-ANEPPS is a dye molecules which has been used to probe membrane electric potentials by its electrochromic response. The dye has an unusual blue shift in the absorption spectrum upon partitioning to lipid membranes. We studied its binding properties to membranes by absorption, fluorescence and resonance Raman spectroscopy. The latter technique provided evidence for the differential solubilization of the dye in the membrane, where the polar head of the molecule protrudes out of the lipid environment and is responsible for the spectral behavior.     

Multiscale molecular dynamics simulations of membrane remodeling by Bin/Amphiphysin/Rvs family proteins

Membrane curvature is no longer thought of as a passive property of the membrane; rather, it is considered as an active, regulated state that serves various purposes in the cell such as between cells and organelle definition. While transport is usually mediated by tiny membrane bubbles known as vesicles or membrane tubules, such communication requires complex interplay between the lipid bilayers and cytosolic proteins such as members of the Bin/Amphiphysin/Rvs(BAR) superfamily of proteins. With rapid developments in novel experimental techniques, membrane remodeling has become a rapidly emerging new field in recent years. Molecular dynamics(MD) simulations are important tools for obtaining atomistic information regarding the structural and dynamic aspects of biological systems and for understanding the physics-related aspects. The availability of more sophisticated experimental data poses challenges to the theoretical community for developing novel theoretical and computational techniques that can be used to better interpret the experimental results to obtain further functional insights. In this review, we summarize the general mechanisms underlying membrane remodeling controlled or mediated by proteins. While studies combining experiments and molecular dynamics simulations recall existing mechanistic models, concurrently, they extend the role of different BAR domain proteins during membrane remodeling processes. We review these recent findings, focusing on how multiscale molecular dynamics simulations aid in understanding the physical basis of BAR domain proteins, as a representative of membrane-remodeling proteins.     

Contrast inversion of the h-BN nanomesh investigated by nc-AFM and Kelvin probe force microscopy

Single sheets of hexagonal boron nitride (h-BN) on transition metals provide a model system for layered insulating materials as well as a functional substrate for molecules and metal clusters. The progress in the understanding of h-BN layers on transition metals was mainly driven by scanning tunnelling microscopy (STM) and photoelectron spectroscopy (PES) measurements within the last decade, while direct measurements of mechanical and electrical properties are still rare. Our investigations of the two-dimensional (2D) h-BN nanomesh on a Rh(111) substrate by high-resolution noncontact atomic force microscopy (nc-AFM) reveal a complex surface structure including a frequently observed contrast inversion. Detailed 2D force spectroscopy measurements are revealing towards a mechanical elastic deformation of the h-BN monolayer caused by the tip-sample interaction. Furthermore, Kelvin probe force microscopy (KPFM) and spectroscopy measurements show local work function variations of the nanomesh, proving the results obtained by PES but additionally providing detailed local information.     

Quantitative imaging of electrospun fibers by PeakForce Quantitative NanoMechanics atomic force microscopy using etched scanning probes

Electrospun polymeric submicron and nanofibers can be used as tissue engineering scaffolds in regenerative medicine. In physiological conditions fibers are subjected to stresses and strains from the surrounding biological environment. Such stresses can cause permanent deformation or even failure to their structure. Therefore, there is a growing necessity to characterize their mechanical properties, especially at the nanoscale.Atomic force microscopy is a powerful tool for the visualization and probing of selected mechanical properties of materials in biomedical sciences. Image resolution of atomic force microscopy techniques depends on the equipment quality and shape of the scanning probe. The probe radius and aspect ratio has huge impact on the quality of measurement.In the presented work the nanomechanical properties of four different polymer based electrospun fibers were tested using PeakForce Quantitative NanoMechanics atomic force microscopy, with standard and modified scanning probes. Standard, commercially available probes have been modified by etching using focused ion beam (FIB). Results have shown that modified probes can be used for mechanical properties mapping of biomaterial in the nanoscale, and generate nanomechanical information where conventional tips fail.     

Curvature-driven lipid sorting in a membrane tubule

Motivated by recent experiments that implicate the mechanical properties of membranes in lipid sorting, we examine the interplay of lipid composition and curvature in membrane tubules. We study how the coupling between membrane composition and membrane bending stiffness and tension affects tubule formation. Drawing a tubule from a vesicle leads to a rearrangement of composition in which the phase of higher flexibility segregates into the highly curved tubule. For point forcing, the force vs extension curve can have a sharp drop just as the tubule begins to form.     

Fabricated cantilever for AFM measurements and manipulations: Pre-stress analysis of stress fibers

The atomic force microscope (AFM) is a highly successful instrument for imaging of nanometer-sized samples and measurement of pico- to nano-Newton forces acting between atoms and molecules, especially in liquid. Generally, commercial AFM cantilevers, which have a sharp tip, are used for AFM experiments. In this review, we introduce micro-fabricated AFM cantilevers and show several applications for cell biology. In manipulation of samples on a cellular scale with a force of tens to hundreds of nano-Newtons, attempts have been made to secure the formation of covalent/non-covalent linkages between the AFM probe and the sample surface. However, present chemistry-based modification protocols of cantilevers do not produce strong enough bonds. To measure the tensile strength and other mechanical properties of actin-based thin filaments in both living and semi-intact fibroblast cells, we fabricated a probe with a hooking function by focused ion beam technology and used it to capture, pull and eventually break a chosen thin filament, which was made visible through fusion with fluorescent proteins. Furthermore, we fabricated a microscoop cantilever specifically designed for pulling a microbead attached to a cell. The microscoop cantilevers can realize high-throughput measurements of cell stiffness.