Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2α. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2α but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of K_d ∼16 μM and Hill coefficient of n ∼2.     

Quantitative determination of the lectin binding capacity of small intestinal brush-border membrane. An enzyme linked lectin sorbent assay (ELLSA)

A test to determine quantitatively the lectin binding sites in brush-border membranes has been developed. Highly purified bovine small intestinal brush-border membranes were prepared, and subsequently coated directly to the bottom of a microtiter plate. Soybean agglutinin conjugated with peroxidase was coupled to its binding sites in the brush-border membranes and the peroxidase activity was determined in a spectrophotometer. The number of soybean agglutinin binding sites in the brush-border membranes has been established by means of iterized computer fit analysis of the data, indicating values for maximal binding of 7.10(-7) M soybean agglutinin per mg of brush-border membrane protein and a dissociation constant of 1.5.10(-5) M.     

Control of erythrocyte membrane microviscosity by insulin

The human erythrocyte membrane binds insulin through high-affinity, low-capacity binding sites (dissociation constant Kd1 2.45 X 10(-9)M; capacity n1 207 fmol/mg protein) and low-affinity, high-capacity binding sites (Kd2 0.63 X 10(-6) M; n2 37 pmol/mg protein). Treatment of the erythrocyte membrane or the intact cells with the physiological concentration of insulin, which is within the range of Kd value of the high-affinity sites, results in a significant reduction of the membrane microviscosity and the filtration time of the intact cells. Use of supraphysiological concentrations of the hormone reverses the effect of the lower concentration of insulin on the membrane microviscosity and the filtration time.     

High-sensitivity cytofluorometric quantitation of lectin and hormone binding to surfaces of living cells.

With a specially equipped flow cytofluorometer it is possible to determine quickly and accurately binding constants and the maximum number of binding sites for ligands such as peptide hormones and lectins on surfaces of intact living cells, with incubation concentrations as low as 10^?11 M. Since the measurement is confined to cell-bound material the cells can be kept in their physiological environment, including free ligand molecules, even at the very moment of the assay. Thus there is no additional risk of perturbing the integrity of the membrane or of interfering with ligand-receptor interactions by washing or similar procedures. It was found that damaged cells, inevitably present in any population, are able to grossly distort binding patterns. Suggestions are given how such cells may be excluded from the measurement.     

Inferring Diffusion Dynamics from FCS in Heterogeneous Nuclear Environments

Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show—first using synthetic data—that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell’s nucleus as well as 2) in the cell’s cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins.     

Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver

The binding of human 125I-labeled HDL3 (high-density lipoproteins, rho 1.125-1.210 g/cm3) to a crude membrane fraction prepared from bovine liver closely fit the paradigm expected of a ligand binding to a single class of identical and independent sites, as demonstrated by computer-assisted binding analysis. The dissociation constant (Kd), at both 37 and 4 degrees C, was 2.9 micrograms protein/ml (approx. 2.9 X 10(-8) M); the capacity of the binding sites was 490 ng HDL3 (approx. 4.9 pmol) per mg membrane protein at 37 degrees C and 115 at 4 degrees C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites (Kd = 41 micrograms protein/ml, approx. 6.7 X 10(-8) M for LDL, and Kd = 5.7 micrograms protein/ml, approx. 7.0 X 10(-9) M for VLDL), but this observation must be considered in light of the fact that the normal circulating concentrations of these lipoproteins are much lower than those of HDL. The binding of 125I-labeled HDL3 to these sites was inhibited only slightly by 1 M NaCl, suggesting the presence of primarily hydrophobic interactions at the recognition site. The binding was not dependent on divalent cations and was not displaceable by heparin; the binding sites were sensitive to both trypsin and pronase. Of exceptional note was the finding that various subclasses of human HDL (including subclasses of immunoaffinity-isolated HDL) displaced 125I-labeled HDL3 from the hepatic HDL binding sites with different apparent affinities, indicating that these sites are capable of recognizing highly specific structural features of ligands. In particular, apolipoprotein A-I-containing lipoproteins with prebeta electrophoretic mobility bound to these sites with a strikingly lower affinity (Kd = 130 micrograms protein/ml) than did the other subclasses of HDL.     

beta-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

Existence of high- and low-affinity vanadate-binding sites on Ca(2+)-ATPase of the sarcoplasmic reticulum.

The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca(2+)-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1 microM, and the low-affinity sites one of 36 microM. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca(2+)-ATPase. These results indicate that 1 mol of Ca(2+)-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxy-ethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13 microM. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca(2+)-ATPase. Vanadate binding to the solubilized Ca(2+)-ATPase was competitively inhibited by inorganic phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detecting ligand interactions with G protein-coupled receptors in real-time on living cells

G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand–GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12 h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs.     

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process

1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the filtration method. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant Kd of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.     

The Kinetics of Cooperative Cofilin Binding Reveals Two States of the Cofilin-Actin Filament

The interaction of cofilin with actin filaments displays positive cooperativity. The equilibrium binding and associated thermodynamic properties of this interaction are well described by a simple, one-dimensional Ising model with nearest neighbor interactions. Here we evaluate the kinetic contributions to cooperative binding and the ability of this model to account for binding across a wide range of cofilin concentrations. A Monte Carlo-based simulation protocol that allows for nearest-neighbor interactions between adjacent binding sites was used to globally fit time courses of human cofilin binding to human nonmuscle (β-, γ-) actin filaments. Several extensions of the one-dimensional Ising model were tested, and a mechanism that includes isomerization of the actin filament was found to best account for time courses of association as well as irreversible dissociation from a saturated filament. This model predicts two equilibrium states of the cofilin-actin, or cofilactin, filament, and the resulting set of binding parameters are in agreement with equilibrium thermodynamic parameters. We conclude that despite its simplicity, this one-dimensional Ising model is a reliable model for analyzing and interpreting the energetics and kinetics of cooperative cofilin-actin filament interactions. The model predicts that severing activity associated with boundaries between bare and decorated segments will not be linear, but display a transient burst at short times on cofilin activation then dissipate due to a kinetic competition between severing activity and cofilin binding. A second peak of severing activity is predicted to arise from irreversible cofilin dissociation on inactivation. These behaviors predict what we believe to be novel mechanisms of cofilin severing and spatial regulation of actin filament turnover in cells. The methods developed for this system are generally applicable to the kinetic analysis of cooperative ligand binding to linear polymers.     

β-Adrenergic receptors in innervated and denervated skeletal muscle

In order to determine if the development of β-adrenergic receptors may explain the catecholamine evoked contracture of denervated mammalian skeletal muscle, the binding capacities and dissociation constants of β-adrenergic receptors of innervated and denervated rat skeletal muscle membrane preparations were determined by using [³H] dihydroalprenolol. The dissociation constants of [³H] dihydroalprenolol binding to innervated and denervated muscle microsomal suspensions were similar. The maximal number of binding sites increased from 27 pmol/g protein to 85 pmol/g protein following 25 days denervation. These results suggest that motor nerve may be involved in part, in the regulation of β-adrenergic receptors in skeletal muscle membrane preparations.     

Competitive Interactions of Ligands and Macromolecular Crowders with Maltose Binding Protein

Cellular signaling involves a cascade of recognition events occurring in a complex environment with high concentrations of proteins, polysaccharides, and other macromolecules. The influence of macromolecular crowders on protein binding affinity through hard-core repulsion is well studied, and possible contributions of protein-crowder soft attraction have been implicated recently. Here we present direct evidence for weak association of maltose binding protein (MBP) with a polysaccharide crowder Ficoll, and that this association effectively competes with the binding of the natural ligand, maltose. Titration data over wide ranges of maltose and Ficoll concentrations fit well with a three-state competitive binding model. Broadening of MBP ¹H­¹⁵N TROSY spectra by the addition of Ficoll indicates weak protein-crowder association, and subsequent recovery of sharp NMR peaks upon addition of maltose indicates that the interactions of the crowder and the ligand with MBP are competitive. We hypothesize that, in the Escherichia coli periplasm, the competitive interactions of polysaccharides and maltose with MBP could allow MBP to shuttle between the peptidoglycan attached to the outer membrane and the ATP-binding cassette transporter in the inner membrane.     

The Hill analysis and co-ion–driven transporter kinetics

ConclusionIn mechanistic models for binding of multiple ligands to a biological unit, the saturation behavior depends on the experimental readout. In the binding model, the Hill analysis does not provide information on the number of binding sites N. In contrast, the Hill analysis of the response model does contain this information, in which case the slope of the curve in the lower concentration range corresponds to the number of binding sites. In neither model does the Hill coefficient—defined as the slope of the curve at half-maximal saturation—report this number. In the binding model, the Hill coefficient varies between a value of 1 in the absence of interaction and a value of N in case of extremely strong interaction. In the response model, it varies between a number larger than 1 and N. In both models, the derived Hill coefficient is a measure of the cooperativity and sets a lowest possible number of sites.Ion-coupled transporters are of the response model type, and the saturation behavior of the rate with the co-ion in the lower concentration limit contains the information on the number of cotransported ions. Additionally, in the case of an ordered-binding mechanism, in which the co-ions bind before the transported substrate, the Hill coefficient of co-ion binding is a function of the substrate concentration. The apparent interaction between the co-ion sites increases with the substrate concentration and, consequently, the Hill coefficient extrapolates to the number of co-ions. Measurements of the Hill coefficient over the entire range of substrate concentrations provide information on both the extent of interaction between the sites and the number of sites.

Online supplemental material

Five supplemental texts accompany this review: (1) derivation of the equations for the binding and response models; (2) derivation of the equations for the ordered-binding transporter mechanism; (3) derivation of the equations for the Hill analysis of the saturation level functions; (4) derivation of the equations for substrate-dependent kinetics of mechanistic transporter models; (5) data analysis by curve fitting. The online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.201411332/DC1.     

Characterization of the interaction between gastrin and its receptor in rat oxyntic gland mucosa

A gastrin receptor, identified in crude membrane preparations of rat oxyntic gland mucosa, has an equilibrium dissociation constant (Kd) of approx. 4 . 10(-10)M and a binding capacity of 4 fmol/mg protein. The binding capacity was significantly lower after 2 days of fasting, parallel with a significant drop in serum gastrin levels; there was no change in Kd. In order to verify Scatchard analysis and to determine if there was a coincident alteration in the association (k+1) and dissociation (k-1) rates in the fasted rat, a kinetics study was performed. Under our conditions, there appeared to be a single set of binding sites and the binding reaction obeyed first-order dissociation, and second-order association rate kinetics. Second-order association rate kinetics were validated by demonstrating the independence of the rate constants when there were alterations in the concentrations of reactants. The average k+1 was determined to be 2 . 10(6) M-1 . s-1. The average k-1 was determined to be 1 . 10(-3) s-1. There was no significant change in the k+1 and k-1 in fed and fasted rats. Fasting decreased the number of gastrin receptors without altering the affinity of the receptor for the hormone.     

Protein�Cprotein binding affinities by pulse proteolysis: Application to TEM-1/BLIP protein complexes

Efficient methods for quantifying dissociation constants have become increasingly important for high‐throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein–ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein–protein complex involving the β‐lactamase TEM‐1 and various β‐lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C_m, of TEM‐1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein–protein complexes. From a small set (n = 4) of TEM‐1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC_m was observed. From this “calibration curve,” accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis‐derived ΔC_m values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high‐throughput mutagenesis binding studies.     

Purification of the 200 kDa hemocyte membrane protein of Sarcophaga peregrina and its specific interaction with fat body

A 200 kDa protein specifically expressed on the surface of pupal hemocytes of Sarcophaga peregrina was purified from the hemocyte membrane. This protein has been suggested to participate in dissociation of the fat body in the pupal stage of this insect. This protein was found to inhibit the dissociation of the fat body in vitro. Furthermore, it was shown to bind to the fat body and the binding could be saturated. These results suggested that pupal hemocytes expressing the 200 kDa protein interact directly with specific binding sites on the basement membrane of the fat body when they disintegrate this tissue.     

Improved high-affinity binding of 3H-apomorphine with a subcellular membrane fraction of mammalian brain tissue

The binding of low concentrations of [³H](?)apomorphine to preparations of calf and rat forebrain tissue was evaluated. Fractionation of crude homogenates to prepare a membrane fraction (P₄) of striatal or caudate homogenates increased the proportion of saturable to total binding from 33% to over 80%, and increased the apparent density of binding sites from 94 to 681 fmol/mg protein. Binding in calf caudate P₄ tissue was protein-dependent and optimal at pH = 7.0 to 7.5, and T = 20 to 25°C; at higher temperatures tissue binding sites appeared to degrade. The half-time of association and dissociation at 22°C were, respectively, 14.0 and 18.5 min; equilibration was complete in 60 min. Kinetic characteristics of high-affinity binding obtained from association and dissociation constants and from saturation isotherms were similar (K_d = 2.1 to 3.4 nM). The pharmacology of competition for ³H-APO suggests selectivity for dopamine-agonist interactions. These results indicate that the P₄ membrane preparation may be useful for the evaluation of dopamine-agonist binding sites or “receptors.”     

Radiation inactivation of binding sites for high density lipoproteins in human fibroblast membranes

Radiation inactivation and target analysis were used to determine the molecular mass of the binding sites for high density lipoproteins (HDL) on membranes prepared from human fibroblasts. These membrane binding sites shared characteristics with the previously described HDL binding sites on whole fibroblasts in tissue culture. They exhibited the same affinity for HDL, the same ligand specificity, and the same sensitivity to proteolytic agents. They were also up-regulated by cholesterol loading of the cells. Kinetics of HDL dissociation from membrane binding sites could not be described by a single exponential function, indicating that HDL probably bind to multiple classes of sites on fibroblast membranes. After exposure to ionizing radiation, these sites decreased in number as an apparent single exponential function of radiation dose, corresponding to an average molecular mass of 16,000 +/- 1,000 Da, which is smaller than any known cell-surface receptor protein. These data indicate that HDL binding sites on fibroblast membranes are not "classical" receptors in that they are kinetically heterogeneous and small in molecular mass.