Characterization of the Resting Axolemma in the Giant Axon of the Squid

Previous electron microscope studies have shown that the Schwann cell layer is traversed by long and tortuous slit-like channels ~60Å wide, which provide the major route of access to the axolemma surface. In the present work the restriction offered by the resting axolemma to the passage of six small non-electrolyte molecules has been determined. The radii of the probing molecules were estimated from constructed molecular models. The ability of the axolemma to discriminate between the solvent (water) and each probing molecule was expressed in terms of the reflection coefficient σ. σ was then used to calculate an effective pore size for the resting axolemma. The value of 4.25 Å found for the pore radius is in excellent agreement with the 1.5 to 8.5 Å limiting values previously calculated from our measurements of water fluxes. The presence of pores with 4.25 Å radius in the resting axolemma is compatible with restricted diffusion of Na. The present paper leads to the conclusion that the axolemma is the only continuous barrier across which the ionic gradient responsible for the normal functioning of the nerve can be maintained. The combined findings of electron microscopy, water permeability, and molecular restricted filtration indicate that in all probability the axolemma is the "excitable membrane" of the physiologists.     

Changes in the Permeability of the Salivary Gland Caused by Sympathetic Stimulation and by Catecholamines

The permeability of the submaxillary gland of cats and dogs has been tested by determining the rates at which non-electrolytes penetrate from the plasma into the saliva. Electrical stimulation of the cervical sympathetic trunk or administration of epinephrine or norepinephrine increases the permeability of the gland enabling glucose (molecular radius, MR = 3.5 Å), sucrose (MR = 4.4 Å), raffinose (MR = 5.6 Å), polyglycol 1000 (MR = 7.2 Å), and polyglycol 1540 (MR = 8.1 Å) to penetrate into the saliva from which they are otherwise excluded. Inulin (MR = 14.7 Å) does not enter the saliva under these circumstances. Analysis of the transfer rates suggests that the molecules diffuse through a pore structure permitting free diffusion for molecules with a radius less than 5.7 Å. Close intraarterial injection of C¹⁴-glucose demonstrates that at least part of this permeability is located in the duct system of the gland. Since epinephrine does not enable sucrose to enter the cells of the gland, it appears that penetration from the extracellular space into the saliva occurs by diffusion through intercellular gaps. The characteristics of the permeability allow conclusions as to the localisation and geometry of the ultrastructural change produced.     

Gaugement of the inner space of the apomyoglobin's heme binding site by a single free diffusing proton. I. Proton in the cavity.

Time resolved fluorimetry was employed to monitor the geminate recombination between proton and excited pyranine anion locked, together with less than 30 water molecules, inside the heme binding site of Apomyoglobin (sperm whale). The results were analyzed by a numerical reconstruction of the differential rate equation for time-dependent diffusion controlled reaction with radiating boundaries using N. Agmon's procedure (Huppert, Pines, and Agmon, 1990, J. Opt. Soc. Am. B., 7:1541-1550). The analysis of the curve provided the effective dielectric constant of the proton permeable space in the cavity and the diffusion coefficient of the proton. The electrostatic potential within the cavity was investigated by the equations given by Gilson et al. (1985, J. Mol. Biol., 183:503-516). According to this analysis the dielectric constant of the protein surrounding the site is epsilon prot < or = 6.5. The diffusion coefficient of the proton in the heme binding site of Apomyoglobin-pyranine complex is D = 4 x 10(-5) cm2/s. This value is approximately 50% of the diffusion coefficient of proton in water. The lower value indicates enhanced ordering of water in the cavity, a finding which is corroborated by a large negative enthropy of binding delta S0 = -46.6 cal.mole-1 deg-1. The capacity of a small cavity in a protein to retain a proton had been investigated through the mathematical reconstruction of the dynamics. It has been demonstrated that Coulombic attraction, as large as delta psi of energy coupling membrane, is insufficient to delay a free proton for a time frame comparable to the turnover time of protogenic sites.     

FLUIDITY OF THE SURFACE OF CULTURED MUSCLE FIBERS : Rapid Lateral Diffusion of Marked Surface Antigens

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23°–25°C than at 0°–4°C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1–3 x 10^-9 cm² s^-1 can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.     

Studies of a serum albumin-liposome complex as a model lipoprotein membrane

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicular, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associate with lipid.Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 Å. The same lamellar separation in the positively charged system is 108 Å. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 Å while that for the positively charged lipoprotein system is 91 Å. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer.Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 Å spacing, characteristic of hexagonal packing of the hydrocarbon chains.A classical light scattering technique is to used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose < arabinose < malonamide < glycerol). K⁺ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.     

Estimating the dielectric constant of the channel protein and pore

When modelling biological ion channels using Brownian dynamics (BD) or Poisson–Nernst–Planck theory, the force encountered by permeant ions is calculated by solving Poisson’s equation. Two free parameters needed to solve this equation are the dielectric constant of water in the pore and the dielectric constant of the protein forming the channel. Although these values can in theory be deduced by various methods, they do not give a reliable answer when applied to channel-like geometries that contain charged particles. To determine the appropriate values of the dielectric constants, here we solve the inverse problem. Given the structure of the MthK channel, we attempt to determine the values of the protein and pore dielectric constants that minimize the discrepancies between the experimentally-determined current–voltage curve and the curve obtained from BD simulations. Two different methods have been applied to determine these values. First, we use all possible pairs of the pore dielectric constant of water, ranging from 20 to 80 in steps of 10, and the protein dielectric constant of 2–10 in steps of 2, and compare the simulated results with the experimental values. We find that the best agreement is obtained with experiment when a protein dielectric constant of 2 and a pore water dielectric constant of 60 is used. Second, we employ a learning-based stochastic optimization algorithm to pick out the optimum combination of the two dielectric constants. From the algorithm we obtain an optimum value of 2 for the protein dielectric constant and 64 for the pore dielectric constant.     

TEMPERATURE AND FREQUENCY OF HEART BEAT IN THE COCKROACH

The frequency of pulsation of the intact heart in nymphs (final (?) instar) of Blatta orientalis L. increases with the temperature according to the equation of Arrhenius. The constant µ has typically the same value, within reasonable limits of error, as that (12,200) deduced for other, homologous activities of arthropods where the rate of central nervous discharge is perhaps the controlling element, namely 12,500 ± calories for temperatures 10–38°C. Below a critical temperature of about 10° a change to a higher value of the temperature characteristic occurs, such that µ = 18,100 ±. Exceptionally (one individual) µ = 14,100 ± over the whole range of observed temperature (4.5–28°). The quantitative correspondence of µ for frequency of heart beat in different arthropods adds weight to the conception that this constant may be employed for the recognition of controlling processes.     

Concentration-dependent effects on fully hydrated DNA at terahertz frequencies

Using terahertz time-domain spectroscopy (THz-TDS), the frequency-dependent dielectric constant of deoxyribonucleic acid (DNA) in solution was measured. The response of the buffer solution is dominated by two Debye modes in this frequency range, and, from an analysis of the concentration dependence, the presence of the DNA increases the main relaxation time and dielectric constant. This reflects the fact that the water in the hydration layer is more tightly bound under the influence of the DNA molecule in comparison to bulk water. This dynamical slowing down with increasing DNA concentration is similar to what is observed with purine nucleotides, but opposite to the behavior of pyrimidine nucleotides. In addition, a suspension model was used with the concentration-dependent data to isolate the dielectric response of the hydrated DNA molecule. The data for the hydrated DNA molecule is still dominated by a Debye response. It is also possible to determine the thickness of the hydration layer, and the DNA molecule influences the surrounding water out to 16 or 17 Å, which corresponds to about six effective hydration layers.     

Kinetics of Diffusion and Convection in 3.2-Å Pores: Exact Solution by Computer Simulation

The kinetics of transport in pores the size postulated for cell membranes has been investigated by direct computer simulation (molecular dynamics). The simulated pore is 11 Å long and 3.2 Å in radius, and the water molecules are modeled by hard, smooth spheres, 1 Å in radius. The balls are given an initial set of positions and velocities (with an average temperature of 313° K) and the computer then calculates their exact paths through the pore. Two different conditions were used at the ends of the pore. In one, the ends are closed and the balls are completely isolated. In the other, the ball density in each end region is fixed so that a pressure difference can be established and a net convective flow produced. The following values were directly measured in the simulated experiments: net and diffusive (oneway) flux; pressure, temperature, and diffusion coefficients in the pore; area available for diffusion; probability distribution of ball positions in the pore; and the interaction between diffusion and convection. The density, viscosity, and diffusion coefficients in the bulk fluid were determined from the theory of hard sphere dense gases. From these values, the “equivalent” pore radius (determined by the same procedure that is used for cell membranes) was computed and compared with the physical pore radius of the simulated pore.     

An Investigation of the Bound Water in Tendon by Dielectric Measurement

The dielectric constant of natural tendon in the frequency range of 200 Hz to 100 kHz has been determined as a function of temperature (300-450°K) for water concentrations ranging from about 6 to 16% by weight. The results compare well with the approach taken by Haggis and his coworkers. Based on the assumption that at low concentration of water the probability of water-water bonding is small and hence may be disregarded, a structure for water in the collagen matrix is proposed in which the water is either bonded in one of four possible states to the polar groups of the polypeptide chains, or is unbound. In determining the distribution of the water among these states an approach similar to that of Haggis and his co-workers, in conjunction with the order-disorder theory of Bragg and Williams, is used. The number of water molecules per unit volume is then determined experimentally by relating it to weight loss as a function of temperature, as determined by thermogravimetric analysis. The dispersion which is normally found in dipolar substances has not been found for tendon. A maximum in the value of the dielectric constant is observed to occur between 25 and 80°C, the temperature depending upon the heating rate.     

Mathematical analysis of the dependence of cell potential on external potassium in corn roots

The K⁺ dependence of normal (ψ) and diffusion (ψ_D) potentials in corn roots [Zea mays L., hybrid (A619 × Oh43) × A632] was determined experimentally and analyzed with respect to the parameter ξ [defined as exp (F ψ/RT)]. In the presence of 10 micromolar carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), ψ behaved as expected of a diffusion potential. Based upon the assumptions (a) that FCCP did not change any term of the Goldman-Hodgkin-Katz equation, and (b) that total potential was functionally the algebraic sum of ψ_D and ψ_P (the deviation from ψ_D due to an electrogenic system), ψ_P was found to be a complex function of external potassium and to have a minimum value of 0.69 millimolar K ion activity outside the cell. Analysis of ψ allowed us to develop an equation which predicts a complicated K⁺ dependence of ψ such as that found by Mertz and Higinbotham (Membrane Transport in Plants and Plant Organelles. Springer-Verlag 1974).     

ON THE TEMPERATURE CHARACTERISTICS FOR FREQUENCY OF BREATHING MOVEMENTS IN INBRED STRAINS OF MICE AND IN THEIR HYBRID OFFSPRING. I

Young mice of a selected line of the dilute brown strain of mice exhibit over the range 15–25°C. (body temperature) a relation of frequency of breathing movements to temperature such that when fitted by the Arrhenius equation the data give a value for the constant µ of 24,000± calories or, less frequently, 28,000±. Young mice of an inbred albino strain show over the range 15–20°C. a value of µ = 34,000±, or, less frequently, 14,000±, with a critical temperature at about 20°C. and a value of µ = 14,000± above 20°C. The F₁ hybrids of these two strains, and the backcross generations to either parent strain, exhibit only those four values of the temperature characteristic observed in the parent strains and none other. One may therefore speak of the inheritance of the value of the constant µ, but the inheritance shows in this instance no Mendelian behavior. Furthermore there appears to be inherited the occurrence (or absence) of a critical temperature at 20°C. These experiments indicate the "biological reality" of the temperature characteristics.     

THE EFFECT OF TEMPERATURE ON THE MECHANICAL ACTIVITY OF THE GILLS OF THE OYSTER (OSTREA VIRGINICA Gm.)

1. The method is described whereby the rate of flow produced by the gills of the oyster can be measured accurately. 2. The rate of doing work in maintaining a constant current along the glass tube can be expressed by the formula W = 2πlµ S ², where W = ergs/sec., l = length of the tube, µ = viscosity in poises, and S = speed at the axis of the tube. 3. The relationship between the rate of doing work and the temperature cannot be described by the equation of Arrhenius. 4. The optimum temperature for the mechanical activity of the gills lies between 25° and 30°C. Below 5° no current is produced, though the cilia are beating. Ciliary motion stops entirely at the freezing temperature of sea water. 5. The factors responsible for the production of current are discussed. The study of the relations between the variability of the rate of flow and the temperature shows that between 15° and 25°C. the absolute variability remains constant and increases considerably above 25° and below 15°. The rôle of the coordination in the production of current is discussed, and the conclusion is reached that coordination is affected by the changes in temperature.     

Characterization of cholesterol oxidase activity in AOT-isooctane reverse micelles and its dependence on micelle size

Summary Characterization of cholesterol oxidase in AOT reverse micelles was performed. pH and temperature profiles show that the entrapment of the enzyme does not change its characteristics appreciably. The enzyme tends to behave as it does in water when micelle size increases and does not maximum rate at some intermediate micelle size. Km was 55–60 fold that in waterAbbreviations and parameters AOT Dioctyl sodium sulfosuccinate - CTAB Cetyl trimethylamonium bromide - K1^E Equilibrium constant of the enzyme between free and bound water - K2^E Equilibrium constant of the enzyme between bound water and surfactant - kf Catalytic constant in free water - kb Catalytic constant in bound water - ks Catalytic constant in surfactant - n Number of water molecules strongly bound to one surfactant molecule     

Bending Free Energy from Simulation: Correspondence of Planar and Inverse Hexagonal Lipid Phases

Simulations of two distinct systems, one a planar bilayer, the other the inverse hexagonal phase, indicate consistent mechanical properties and curvature preferences, with single DOPE leaflets having a spontaneous curvature, R₀ = −26 Å (experimentally ∼–29.2 Å) and DOPC leaflets preferring to be approximately flat (R₀= –65 Å, experimentally ∼–87.3 Å). Additionally, a well-defined pivotal plane, where a DOPE leaflet bends at constant area, has been determined to be near the glycerol region of the lipid, consistent with the experimentally predicted plane. By examining the curvature frustration of both high and low curvature, the transferability of experimentally determined bending constants is supported. The techniques herein can be applied to predict the effect of biologically active molecules on the mechanical properties of lipid bilayers under well-controlled conditions.     

Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells

Phosphatidylinositol 4,5-bisphosphate (PIP₂) controls a surprisingly large number of processes in cells. Thus, many investigators have suggested that there might be different pools of PIP₂ on the inner leaflet of the plasma membrane. If a significant fraction of PIP₂ is bound electrostatically to unstructured clusters of basic residues on membrane proteins, the PIP₂ diffusion constant, D, should be reduced. We microinjected micelles of Bodipy TMR-PIP₂ into cells, and we measured D on the inner leaflet of fibroblasts and epithelial cells by using fluorescence correlation spectroscopy. The average ± SD value from all cell types was D = 0.8 ± 0.2 μm²/s (n = 218; 25°C). This is threefold lower than the D in blebs formed on Rat1 cells, D = 2.5 ± 0.8 μm²/s (n = 26). It is also significantly lower than the D in the outer leaflet or in giant unilamellar vesicles and the diffusion coefficient for other lipids on the inner leaflet of these cell membranes. The simplest interpretation is that approximately two thirds of the PIP₂ on inner leaflet of these plasma membranes is bound reversibly.     

Quantifying Interactions of a Membrane Protein Embedded in a Lipid Nanodisc using Fluorescence Correlation Spectroscopy

Using fluorescence correlation spectroscopy, we measured a dissociation constant of 20 nM between EGFP-labeled LcrV from Yersinia pestis and its cognate membrane-bound protein YopB inserted into a lipid nanodisc. The combination of fluorescence correlation spectroscopy and nanodisc technologies provides a powerful approach to accurately measure binding constants of interactions between membrane bound and soluble proteins in solution. Straightforward sample preparation, acquisition, and analysis procedures make this combined technology attractive for accurately measuring binding kinetics for this important class of protein-protein interactions.Interactions involving membrane proteins are integral to a multitude of cellular processes, including signal transduction, energy production and conversion, cell adhesion, and foreign molecule identification. More than half of all pharmaceutical drugs target membrane proteins, further illustrating their importance in human health (1). Due to this, there is a continuing high demand for methods that can screen, validate, and quantify interactions involving membrane proteins. Unfortunately, the quantitative techniques available to characterize protein-protein interactions are most often directed toward soluble proteins, and are often difficult to apply to membrane proteins (2). Recently, advances in lipid nanodisc technologies, often referred to as reconstituted high-density lipoproteins, have enabled biophysical and biochemical studies of solubilized membrane proteins. The nativelike lipid environment of nanodiscs maintains protein functionality, opening a realm of possibilities in analyzing membrane protein function and dynamics in solution (3).Nanodiscs are discoidal cell membrane mimetics that are 8–20 nm in diameter, consisting of a lipid bilayer stabilized by two peripheral apolipoprotein A-I proteins (4). These particles provide an excellent alternative to traditional lipid-based platforms (e.g., liposomes) for membrane protein solubilization and interrogation. The diameter of nanodisc can be engineered to accommodate different-sized membrane proteins by varying lipid composition. As such, nanodiscs represent an important platform for expression, isolation, and study of functional membrane proteins and the multiprotein complexes they form. Several groups have successfully reconstituted a variety of membrane proteins, including bacteriorhodopsin (5), G-protein coupled receptors (6), and cytochrome P450 (7) to name a few. To produce solubilized, discrete membrane proteins, we utilized a cell-free expression approach to embed membrane proteins directly into nanodiscs formed in situ. This approach allows for enhanced purification and rapid labeling of proteins of interest where traditional approaches are unsuccessful (8). Fluorescence correlation spectroscopy (FCS) analysis coupled with nanodisc technology has been successfully used to monitor small ligand binding interactions with membrane proteins (6,8) and to measure lipid-protein interactions at the single molecule level (9). Here, we show that FCS can be used to measure interactions between soluble proteins and cognate membrane proteins inserted into nanodiscs produced using cell-free expression methodologies.FCS uses correlation analysis of fluorescence arising from randomly diffusing molecules to measure diffusion constants, and hence hydrodynamic radii, of species in solution. Diffusion times measured by FCS are inversely proportional to diffusion constants. A shift to slower diffusion time indicates an increase in the hydrodynamic radius due to binding. Due to this size sensitivity, FCS has been proposed repeatedly as a method to quantify molecular interactions in solution (10). However, an important difficulty in FCS arises when characterizing interactions between two proteins of similar size. The diffusion time is inversely proportional to the cube root of the molecular mass τ_D ∼ M^−1/3. Doubling the mass results in only a factor-of-1.3 increase in diffusion time, but a factor of 1.6 is required to resolve two species (11). As we demonstrate, this difficulty is overcome by using FCS to measure the interactions between labeled soluble proteins and membrane proteins supported within a nanodisc. The much larger size of the membrane protein-nanodisc complex (Fig. 1 A) relative to most soluble proteins provides the necessary differential in diffusion times to easily resolve bound and free species by FCS.Open in a separate windowFigure 1(A) Model of YopB (blue) inserted into a 10-nm nanodisc with cognate protein LcrV (red) labeled with EGFP (green). The molecular masses used in this model are: LcrV (35 kDa), EGFP (27 kDa), YopB (42 kDa, monomer), and nanodisc-YopB complex (258 kDa). The small LcrV binds to the much larger YopB-nanodisc complex, resulting in a significant shift in the autocorrelation curves to longer diffusion times. (B) Hypothetical autocorrelation curves expected for a series of titration experiments measured using FCS. Increased binding is measured as an increase in diffusion time. For intermediate binding, diffusion components are detectable as illustrated (red, green, and blue curves). To see this figure in color, go online.Binding kinetics are readily measured using FCS by titrating increasing amounts of unlabeled cognate protein, resulting in a series of autocorrelation curves similar to those modeled in a hypothetical example in Fig. 1 B. For 0% binding (black curve), only the labeled soluble protein is present, producing a single component correlation curve with a diffusion time of 0.1 ms. On the autocorrelation curve with a single component, the diffusion time is the time at which the autocorrelation amplitude decreases by half. For 100% binding (cyan curve), the amount of nanodisc with inserted membrane protein is well above the dissociation constant so that all available soluble protein is bound.Again, a single component correlation curve is found, but now with a longer diffusion time (10 ms in this hypothetical example). Intermediate cases have two components, corresponding to free and bound protein. The relative amplitudes of the components obtained by fitting to two component models in these cases can be used to obtain a binding curve. For binding interactions, the amount of ligand in the free and bound state can be separated by their diffusion time and quantified as a function of concentration. The fraction of bound protein is calculated by fitting the correlation to two components. The first component f₀(τ) is the correlation of the free protein. The second component g₀(τ) is the correlation when the protein is bound. The total correlation function ish(τ) = αf₀(τ) + βg₀(τ), (1)where f₀(τ) = 1/(1 + τ/τ_f) and g₀(τ) = 1/(1 + τ/τ_b). The values α and β are, respectively, the amplitudes of the correlation function for free and bound protein, with the corresponding diffusion times τ_f and τ_b. The ratio F = β/(α + β) gives the fraction bound.We investigated by FCS the interaction of two Yersinia pestis proteins: soluble EGFP-labeled LcrV and membrane-bound YopB (Fig. 1 A). YopB is an effector protein involved in host cell invasion and disarming the cell’s defense. Although the structure of YopB is largely unknown, it is thought to exist as a dimer (12). Delivery of YopB to the host cell is regulated by the Type III secretion system (T3SS) (i.e., injectisome). The needle tip of the injectisome contains the LcrV protein. When Y. pestis comes in contact with a cell membrane, the injectisome forms a pore through the membrane to facilitate the diffusion of effector proteins into the host cell. Multiple lines of evidence suggest that pores are formed only through the direct interaction of YopB with LcrV (13).The FCS diffusion times of free LcrV (τ_f; Fig. 2 A, black curve) and fully bound LcrV (τ_b; [YopB] = 10 μM) were found to be 140 and 630 μs, respectively. Because no measurable change in diffusion time was detected above [YopB] = 1 μM (Fig. 2, green curve), LcrV was assumed to be completely bound at [YopB] = 10 μM. The diffusion times of free and bound LcrV were determined by fitting the autocorrelation curves to a one-component model (χ² ∼ 1 for each diffusion time). These diffusion times correspond to average hydrodynamic radii of 2.5 nm for free LcrV and 11.1 nm for the LcrV + YopB-nanodisc complex, as calculated by the Einstein-Stokes equation. No significant change in diffusion time was detected when LcrV was incubated with 2 μM of nanodiscs lacking the YopB protein (Fig. 2 A, gray curve), suggesting that LcrV alone is unable to interact directly with a lipid bilayer.Open in a separate windowFigure 2(A) FCS autocorrelation curves of 1 nM EGFP-labeled LcrV protein in the absence and presence of YopB at increasing concentrations. The diffusion time of freely diffusing LcrV is τ_D,free = 140 s (black curve). At [YopB] = 1 μM where LcrV is completely bound, the FCS curve is dominated by a single component (green curve). The autocorrelation curves at intermediate concentrations (in particular [YopB] = 25 and 50 nM, blue and cyan curves) contain contributions from two diffusing components. A control experiment was performed with 2 μM of nanodisc lacking the YopB protein (gray curve). No significant change in diffusion time was detected. (B) The fraction of bound LcrV as a function of YopB concentration (log scale). This fraction is calculated by fitting the FCS curves shown in Fig. 2A with Eq. 1. The dissociation constant extracted is K_d = 20.45 ± 2.0 nM. Error bars represent an average over six FCS measurements. To see this figure in color, go online.To extract the dissociation constant (K_D), a titration was performed over a range of YopB concentrations (10 pM to 1 μM), obtained by varying the concentration of YopB-nanodisc complexed. Having determined the values of the diffusion times for free (τ_f) and bound (τ_b) protein, we fitted the autocorrelation curves to a two-species correlation function with Eq. 1. The brightness per LcrV molecule was between 2170 and 2280 photons/(s × molecule) for the entire titration, validating the use of Eq. 1. The only varying parameters were the correlation amplitudes α (free protein) and β (bound protein). Intermediate binding was observed between 10 and 100 nM, because two correlation components are resolved. At [YopB] = 50 nM (cyan curve), the ratio β/α was 1.5, indicating that more than half of LcrV was bound. For the two-component model in these intermediate cases, the χ² value was between 1 and 1.3, indicating a good fit. In contrast, fitting the data to a one-component model resulted in a poor fit (χ² > 7), justifying the use of the two-species model. Binding curves were fitted to the equation y = x/(K_D + x), where y is the fraction of bound LcrV, x is the YopB concentration, and K_d the dissociation constant.Fitting this model with a least-squares algorithm (Fig. 2 B), we extracted K_d = 20.5 ± 2.0 nM. To quantify the statistical error, three measurements were recorded for 2 min, and the entire titration was repeated. Importantly, these data provide not only a quantitative binding affinity for the YopB-LcrV interaction, but support the hypothesis that LcrV requires direct interaction with YopB, not just a lipid bilayer, to promote pore formation.In conclusion, the coupling of FCS with nanodisc technology provides a facile yet powerful tool to quantitatively measure interactions involving membrane proteins in solution. FCS can simultaneously detect the presence of both free and bound species without the need for surface immobilization of the cognate proteins. Cell-free coexpression of both membrane protein and apolipoprotein components in the presence of lipids is a facile methodology for producing functional, soluble, nanodisc-supported membrane proteins. Labeling the soluble cognate proteins with EGFP provides a simple path to obtaining fluorescent, single-labeled proteins compatible with FCS. The combination of FCS with nanodisc technology presented here provides not only new key data for modeling the invasion process of Y. pestis, but can also be generalized to study interactions between most other soluble and membrane proteins. Such methods have been lacking, yet are critical for understanding interaction networks, e.g., signal transduction cascades.See the Supporting Material for additional detail on the methodology used.     

Dynamics of the Serine Chemoreceptor in the Escherichia coli Inner Membrane: A High-Speed Single-Molecule Tracking Study

We investigated the mobility of the polar localized serine chemoreceptor, Tsr, labeled by the fluorescent protein Venus in the inner membrane of live Escherichia coli cells at observation rates up to 1000 Hz. A fraction (7%) of all Tsr molecules shows free diffusion over the entire cell surface with an average diffusion coefficient of 0.40 ± 0.01 μm² s⁻¹. The remaining molecules were found to be ultimately confined in compartments of size 290 ± 15 nm and showed restricted diffusion at an inner barrier found at 170 ± 10 nm. At the shortest length-scales (<170 nm), all Tsr molecules diffuse equally. Disruption of the cytoskeleton and rounding of the cells resulted in an increase in the mobile fraction of Tsr molecules and a fragmenting of the previously polar cluster of Tsr consistent with a curvature-based mechanism of Tsr cluster maintenance.     

KINETOPLAST DEOXYRIBONUCLEIC ACID OF THE HEMOFLAGELLATE TRYPANOSOMA LEWISI

Cesium chloride centrifugation of DNA extracted from cells of blood strain Trypanosoma lewisi revealed a main band, ρ = 1.707, a light satellite, ρ = 1.699, and a heavy satellite, ρ = 1.721. Culture strain T. lewisi DNA comprised only a main band, ρ = 1.711, and a light satellite, ρ = 1.699. DNA isolated from DNase-treated kinetoplast fractions of both the blood and culture strains consisted of only the light satellite DNA. Electron microscope examination of rotary shadowed preparations of lysates revealed that DNA from kinetoplast fractions was mainly in the form of single 0.4 µ circular molecules and large masses of 0.4 µ interlocked circles with which longer, often noncircular molecules were associated. The 0.4 µ circular molecules were mainly in the covalently closed form: they showed a high degree of resistance to thermal denaturation which was lost following sonication; and they banded at a greater density than linear DNA in cesium chloride-ethidium bromide gradients. Interpretation of the large masses of DNA as comprising interlocked covalently closed 0.4 µ circles was supported by the findings that they banded with single circular molecules in cesium chloride-ethidium bromide gradients, and following breakage of some circles by mild sonication, they disappeared and were replaced by molecules made up of low numbers of apparently interlocked 0.4 µ circles. When culture strain cells were grown in the presence of either ethidium bromide or acriflavin, there was a loss of stainable kinetoplast DNA in cytological preparations. There was a parallel loss of light satellite and of circular molecules from DNA extracted from these cells.