Quantitative Microscopy: Protein Dynamics and Membrane Organisation

The mobility of membrane proteins is a critical determinant of their interaction capabilities and protein functions. The heterogeneity of cell membranes imparts different types of motion onto proteins; immobility, random Brownian motion, anomalous sub-diffusion, 'hop' or confined diffusion, or directed flow. Quantifying the motion of proteins therefore enables insights into the lateral organisation of cell membranes, particularly membrane microdomains with high viscosity such as lipid rafts. In this review, we examine the hypotheses and findings of three main techniques for analysing protein dynamics: fluorescence recovery after photobleaching, single particle tracking and fluorescence correlation spectroscopy. These techniques, and the physical models employed in data analysis, have become increasingly sophisticated and provide unprecedented details of the biophysical properties of protein dynamics and membrane domains in cell membranes. Yet despite these advances, there remain significant unknowns in the relationships between cholesterol-dependent lipid microdomains, protein-protein interactions, and the effect of the underlying cytoskeleton. New multi-dimensional microscopy approaches may afford greater temporal and spatial resolution resulting in more accurate quantification of protein and membrane dynamics in live cells.     

Nanogap Dielectric Spectroscopy for Aptamer-Based Protein Detection

Among the various label-free methods for monitoring biomolecular interactions, capacitive sensors stand out due to their simple instrumentation and compatibility with multiplex formats. However, electrode polarization due to ion gradient formation and noise from solution conductance limited early dielectric spectroscopic measurements to high frequencies only, which in turn limited their sensitivity to biomolecular interactions, as the applied excitation signals were too fast for the charged macromolecules to respond. To minimize electrode polarization effects, capacitive sensors with 20 nm electrode separation were fabricated using silicon dioxide sacrificial layer techniques. The nanoscale separation of the capacitive electrodes in the sensor results in an enhanced overlapping of electrical double layers, and apparently a more ordered “ice-like” water structure. Such effects in turn reduce low frequency contributions from bulk sample resistance and from electrode polarization, and thus markedly enhance sensitivity toward biomolecular interactions. Using these nanogap capacitive sensors, highly sensitive, label-free aptamer-based detection of protein molecules is achieved.     

Voltage and Current Clamp Transients with Membrane Dielectric Loss

Transient responses of a space-clamped squid axon membrane to step changes of voltage or current are often approximated by exponential functions of time, corresponding to a series resistance and a membrane capacity of 1.0 μF/cm². Curtis and Cole (1938, J. Gen. Physiol. 21:757) found, however, that the membrane had a constant phase angle impedance z = z₁(jωτ)^-α, with a mean α = 0.85. (α = 1.0 for an ideal capacitor; α < 1.0 may represent dielectric loss.) This result is supported by more recently published experimental data. For comparison with experiments, we have computed functions expressing voltage and current transients with constant phase angle capacitance, a parallel leakage conductance, and a series resistance, at nine values of α from 0.5 to 1.0. A series in powers of t^α provided a good approximation for short times; one in powers of t^-α, for long times; for intermediate times, a rational approximation matching both series for a finite number of terms was used. These computations may help in determining experimental series resistances and parallel leakage conductances from membrane voltage or current clamp data.     

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data.     

Single-Molecule Force Spectroscopy of Membrane Protein Folding

Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically manipulating them over a wide force range. Here, we review the current understanding of membrane protein folding learned by using the force spectroscopy approach. Membrane protein folding in lipid bilayers is one of the most complex biological processes in which diverse lipid molecules and chaperone proteins are intricately involved. The approach of single protein forced unfolding in lipid bilayers has produced important findings and insights into membrane protein folding. This review provides an overview of the forced unfolding approach, including recent achievements and technical advances. Progress in the methods can reveal more interesting cases of membrane protein folding and clarify general mechanisms and principles.     

Relation Between the Dielectric Constant of Hydrophobic Cation Exchange Membrane and Membrane Permeability to Counterions

Filters made of cellulose acetate-nitrate when saturated with organic solvents and interposed between aqueous solutions form membranes which behave like cation exchangers. The diffusion coefficients of counterions in such membranes are strongly dependent upon the dielectric constant of the saturating solvent. The results obtained suggest that a linear relationship between the log of the cation's diffusion coefficient (or membrane conductance) and the reciprocal value of the dielectric constant of the saturating solvent exists. There is also a good correlation between the relative membrane permeability to organic cations and the solubility of the cations in the pure solvent phase. These studies indicate that there are two routes for cation movement through the membrane: (a) the bulk hydrophobic phase and (b) continuous narrow aqueous channels.     

Surface-Enhanced Raman Spectroscopy of the Endothelial Cell Membrane

We applied surface-enhanced Raman spectroscopy (SERS) to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces.     

z-STED Imaging and Spectroscopy to Investigate Nanoscale Membrane Structure and Dynamics

Super-resolution stimulated emission depletion (STED) microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy) or axially (z). Two-dimensional STED has been extensively used to elucidate the nanoscale membrane structure and dynamics via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of ∼100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles, or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively.     

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, ΔG°_H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.Download video file.^{(66M, mov)}     

The Double Fixed Charge Membrane: Low Frequency Dielectric Dispersion

An analysis is made of the AC characteristics of a membrane consisting of two fixed charge regions of opposite sign, in contact. It is shown that the equivalent parallel capacitance and conductance of such a membrane undergo a strong dispersion at low frequencies. The dielectric dispersion is a result of polarization effects in the diffusion of coions in each of the two fixed charge lattices. This, at low frequencies, gives rise to a very large diffusion capacitance. The form of the dispersion characteristics is very similar to those observed for synthetic-fused anion-cation membranes and various cellular membranes.     

Quantitation of pH-induced Aggregation in Binary Protein Mixtures by Dielectric Spectroscopy

This paper presents a quantitative approach for measuring pH-controlled protein aggregation using dielectric spectroscopy. The technique is demonstrated through two aggregation experiments, the first between ??-lactoglobulin (??-Lg) and hen lysozyme (HENL) and the second between bovine serum albumin (BSA) and HENL. In both experiments, the formation of aggregates is strongly dependent on the solution pH and is clearly indicated by a decrease in the measured permittivity when the second protein is added. A quantifiable lower-bound on the ratio of proteins involved in the aggregation process is obtained from the permittivity spectra. Lower-bound aggregation ratios of 83?% for ??-Lg/HENL at pH?6.0 and 48?% for BSA/HENL at pH?9.2 were consistent with turbidity measurements made on the same solutions.     

Measurement of Dipole Potential in Bilayer Lipid Membranes by Dielectric Spectroscopy

Planar bilayer lipid membranes formed from egg phosphatidylcholine in aqueous media containing the lipophilic anion, dipicrylamine (DPA), were studied by dielectric spectroscopy over a frequency range of 10 Hz–10 MHz. The membranes showed dielectric relaxation due to the translocation of DPA between the membrane interfaces. Incorporating either cholesterol or 6-ketocholestanol into the membranes increased the characteristic frequency of the relaxation, which is proportional to the translocation rate constant of DPA. The results suggested that the sterol dipoles induced positive potential changes within the membrane interior. The changes of the dipole potential were 70 mV for cholesterol and 150 mV for 6-ketocholestanol when the sterol mole fraction was 0.67. The opposite effect was caused by phloretin added to the aqueous media, and the maximum dipole potential change was ?90 mV at 100 μM.     

Mitochondrial Membrane Studies Using Impedance Spectroscopy with Parallel pH Monitoring

A biological microelectromechanical system (BioMEMS) device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy) to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1) electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2) ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample’s complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles.     

Dielectric Spectroscopy of Chloroplasts Isolated from Higher Plants--Characterization of the Double-Membrane System

The dielectric dispersion of individual chloroplasts has beenmeasured between 1 kHz and 32 MHz. As measuring techniques,both electrorotation and dielectrophoresis were used. We analysedthe dispersion data on the basis of a "three-shell model", inwhich three concentric shells are taken to represent the outerand inner envelope membranes of the plastid and the intermembranespace. The measurements revealed that the outer and inner envelopemembranes of the chloroplasts were different in terms of theirelectrical properties. Both membranes are visible in the rotationspectra as different relaxation processes under particular conditions.Less information can be obtained about the intermembrane spaceand the thylakoid systems. A set of parameters describing theelectrical properties of plastids can be obtained, however,for each plastid compartment, with different degrees of accuracy.Dielectrophoretic motion of individual chloroplasts was measuredbetween 4 kHz and 32 MHz and the results were in good agreementwith a theoretical analysis. Plots of both types of spectrum(rotational and dielectrophoretic motion) lead to curves analogousto Cole-Cole curves, with semicircles in all quadrants of thecoordinate system. Differences between these results and impedancedata are discussed. (Received October 11, 1989; Accepted July 23, 1990)     

Dielectric Relaxation Dynamics of Water in Model Membranes Probed by Terahertz Spectroscopy

We study hydrated model membranes, consisting of stacked bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids, using terahertz time-domain spectroscopy and infrared spectroscopy. Terahertz spectroscopy enables the investigation of water dynamics, owing to its sensitivity to dielectric relaxation processes associated with water reorientation. By controlling the number of water molecules per lipid molecule in the system, we elucidate how the interplay between the model membrane and water molecules results in different water dynamics. For decreasing hydration levels, we observe the appearance of new types of water dynamics: the collective bulklike dynamics become less pronounced, whereas an increased amount of both very slowly reorienting (i.e., irrotational) and very rapidly reorienting (i.e., fast) water molecules appear. Temperature-dependent measurements reveal the interconversion between the three distinct types of water present in the system.     

Plasmon Spectroscopy for Subnanometric Copper Particles: Dielectric Function and Core–Shell Sizing

In the last years, there has been a growing interest in the study of transition metal nanoparticles (Nps) due to their potential applications in several fields of science and technology. In particular, their optical properties are governed by the characteristics of the dielectric function of the metal, its size and environment. This work analyses the separated contribution of free and bound electrons on the optical properties of copper Nps. Usually, the contribution of free electrons to the dielectric function is corrected for particle size through the modification of the damping constant, which is changed as usual introducing a term inversely proportional to the particle’s radius to account for the extra collisions with the boundary when the size approaches the electronic mean free path limit (about 10 nm). For bound electron contribution, the interband transitions from the d-band to the conduction band are considered together with the fact that the electronic density of states in the conduction band must be made size-dependent to account for the larger spacing between electronic energy levels as the particle decreases in size below 2 nm. Taking into account these specific modifications of free and bound electron contributions to the dielectric function, it was possible to fit the bulk complex dielectric function, and consequently, determine optical parameters and band energy values such as the coefficient for bound electron contribution Q _bulk?=?2?×?10²⁴, gap energy E _g?=?1.95 eV, Fermi energy E _F?=?2.15 eV, and damping constant for bound electrons γ _b?=?1.15?×?10¹⁴ Hz. With both size-dependent contributions to the dielectric function, extinction spectra of copper Nps in the subnanometer radius range can be calculated using Mie’s theory and its behaviour with size can be analysed. These studies are applied to fit experimental extinction spectra of very small spherical core–shell Cu–Cu₂O Nps generated by ultrafast laser ablation of a solid target in water. Theoretical calculations for subnanometric core radius are in excellent agreement with experimental results obtained from core–shell colloidal Nps. From the fitting, it is possible determining core radius and shell thickness of the Nps, showing that optical extinction spectroscopy is a good complementary technique to standard high-resolution electron microscopy for sizing spherical nanometric-subnanometric Nps.