Real-time analysis of antibody interactions with whole enveloped human cytomegalovirus using surface plasmon resonance

Human cytomegalovirus (CMV) is a large enveloped virus that encodes multiple glycoproteins required for virus-cell binding and fusion. To assess the binding properties of antibodies with target glycoprotein in a natural context of infection, we investigated the feasibility of using the surface plasmon resonance (SPR) technique for studying the direct binding of antibodies with CMV virions. Direct immobilization of whole virions to sensor surface and a surface regeneration procedure allowed for quantitative and reproducible measurements of binding affinity and binding kinetics of antibody–whole virion interactions. The conformational and functional integrity of viral particles was not compromised by the regeneration condition as evaluated with antibodies recognizing conformational epitopes and by electron microscopy. Binding of an irrelevant antibody was not observed, indicating the high specificity of the method. A panel of anti-gB antibodies was measured and the binding affinities correlated fairly well with those determined by ELISA. These data demonstrated that the interaction of anti-gB antibody with whole virion of large enveloped CMV can be quantitatively studied using SPR. This method has been successfully applied for screening and selection of anti-CMV antibodies and can be potentially extended to study antibody–glycoprotein interactions of other related herpesviruses.     

Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase

Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC₅₀ values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.     

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from Dictyostelium discoideum plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent $\text{D.}\text{discoideum}$ cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [³H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.     

Efficient removal of detergents from proteins and peptides in a spin column format

Detergents are commonly used in protein–chemistry protocols and may be necessary for protein extraction, solubilization, and denaturation; however, their presence interferes with many downstream analysis techniques, including mass spectrometry (MS). To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. In this study, we describe a high-performance resin that offers exceptional detergent removal for proteins and peptides. When used in a spin column format, this resin dramatically improves protein and peptide MS results by more than 95% removal of 1–5% detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate, Chaps, Triton X-100, Triton X-114, NP-40, Brij-35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Postcolumn liquid chromatography–tandem MS (LC–MS/MS) analysis of trypsin digests of bovine serum albumin (BSA) and HeLa cell lysate revealed excellent sequence coverage, indicating successful removal of detergent from the peptides. Matrix-assisted laser desorption/ionization (MALDI)–MS analysis of unprocessed and processed samples further confirmed efficient removal of detergents. The advantages of this method include speed (<15 min), efficient detergent removal, and high recovery of proteins and peptides.     

The action of a binary nonionic detergent on a kidney membrane fraction.

The disruption of a kidney cortex microsomal membrane preparation by a binary, nonionic detergent, was followed by using as markers, the changes in total protein content, and (Na+, K+)-ATPase in a supernatant fraction. Both markers responded similarly to changes in pH, microsome concentration and detergent concentration, but responded differently for time-dependent studies. The (Na+, K+)-ATPase activity was increased 2.2-fold (76.1 mumoles Pi/mg protein/h, 95% ouabain-sensitive) by a single detergent treatment and 3.5-fold (92% ouabain-sensitive) by a sequential detergent treatment. Changes in the critical micelle concentration (cmc) were observed for varying detergent and protein concentrations, which suggest interactions of monomeric detergent with the membrane. The peak of (Na+, K+)-ATPase activity occurred above the cmc which suggests the participation of micelles in releasing the enzyme from the membranes. Hill plots of the protein released as the detergent concentration was varied showed a change in the slope near the cmc indicating a four-fold increase in the binding of detergent to membranes as the detergent concentration is increased above the cmc. These results suggest that the disruption of membranes by detergent involves the binding of detergent monomers to the membrane followed by the formation of co-micelles of the detergent with segments of the membrane to complete the separation process.     

The leader region of pre-maltose binding protein binds amphiphiles. A model for self-assembly in protein export

Maltose binding protein, like most periplasmic proteins, is resistant to a variety of proteinases. Treatment of pre-maltose binding protein with trypsin, chymotrypsin, or proteinase K removes an amino-terminal domain of the same approximate size as the leader sequence without degrading the mature portion of the protein. In addition, pre-maltose binding protein is as active as mature in binding maltose (Ferenci, T., and Randall, L.L. (1979) J. Biol. Chem. 254, 9979-9981). By these criteria, the precursor and mature proteins are in the same conformation except for the exposed leader sequence on the precursor. We have compared the ability of these proteins to interact with amphipaths, such as detergents. The precursor protein binds to Triton X-100, while the mature protein does not. We propose that the leader domain is responsible for detergent binding. Mutations in the leader region of the precursor which block export in vivo prevent detergent binding in vitro. A mutant with a mild export defect can still bind detergent. This correlation between detergent binding by precursors with related leaders and export efficiency of each precursor suggests that hydrophobic partition of the leader may initiate pre-protein transfer across the membrane.     

Biochemical properties of 9-cis- and all-trans-retinoylopsins

The stoichiometry of the reaction between [14C]-9-cis-retinoyl fluoride, a close isostere of 9-cis-retinal, and bovine opsin and the biochemical and spectral properties of this new pigment were investigated. The stoichiometry of retinoid incorporation is approximately one in dodecyl maltoside, a detergent in which opsin is capable of regeneration with 11-cis-retinal. Interestingly, in Ammonyx LO, a detergent that does not permit rhodopsin regeneration, the stoichiometry of binding is still approximately one. By contrast, heat-denatured opsin does not irreversibly bind substantial [14C]retinoyl fluoride. This result strongly suggests that the nucleophilicity of the active site lysine is retained in Ammonyx LO but that further conformational changes in the protein, required to form rhodopsin, are not possible. These results are all consistent with an active site directed mechanism for the irreversible reaction of 9-cis-retinoyl fluoride with opsin probably at the active site lysine residue. The ultraviolet spectra of 9-cis-retinoylopsin and its all-trans congener show gamma max's at 373 and 380 nm, respectively, somewhat bathochromically shifted from their respective model N-butylretinamides which absorb at 347 and 351 nm. Photolysis of both 9-cis- and all-trans-retinoylopsins leads to the same photostationary state. This shows that, as expected, photoisomerization without bleaching occurs. The photolysis of either 9-cis- or all-trans-retinoylopsin in the presence of the G protein (transducin) does not lead to the activation of the latter. This is consistent with the notion that a protonated Schiff base is critical for the function of rhodopsin.     

Evidence for a phospholipid requirement in the specific binding of glucocorticoids to receptors of fibroblasts and thymic lymphocytes.

The specific steroid binding capacity of soluble preparations from mouse fibroblasts and rat thymic lymphocytes is inactivated by incubation with phospholipases. Receptor binding is drastically reduced by very low concentrations of boiled phospholipase A preparations from bee venom and snake venoms. The enzyme effect is calcium-dependent and is blocked by both phospholipid and a substrate analog that is a competitive inhibitor of phospholipase A. The specific binding capacity is also sensitive to digestion by phospholipase C. Two possible mechanisms are considered for the phospholipase A effect: (a) the receptor protein may be associated with a phospholipid component which is required for specific hormone binding; (b) phospholipase A may be producing detergent products that are indirectly inactivating the receptor. Examination of the effects of lysophosphatide on the receptor and assay of lipid phosphate in the receptor preparation do not support a mechanism based solely on detergent effects. Because phospholipase C, which does not produce detergent products, also inactivates the binding, we propose that the phospholipases may be digesting the phospholipid which is a requisite component of the glucocorticoid receptor.     

Multiple equilibria binding treatment of lipid and detergent interactions with membrane proteins. Application to cytochrome c oxidase solubilized in cholate

A modified multiple binding equilibria treatment is presented that allows determination of thermodynamic parameters of the interaction of phospholipids with integral membrane proteins solubilized in excess detergent. Lipid binding is modeled as a series of exchange reactions between lipid molecules and detergent molecules at the hydrophobic protein surface. A general equation is derived which expresses a relative association constant (K) and the total number of contact sites at the lipid-protein interface (N) in terms of experimentally measurable variables. A useful simplification of the general equation occurs when the amount of detergent is high relative to the total number of lipid binding sites in the sample. Computer simulations show that in cases we have examined there appears to be an experimentally accessible range of detergent to protein molar ratios where the approximation at high detergent is useful for analyzing experimental data. This model is used to examine the competition between cholate and spin-labeled phospholipids for the hydrophobic surfaces of bovine heart cytochrome c oxidase. We find, for example, that K = 12 +/- 2 for phosphatidylcholine relative to cholate (i.e., the cholate molecules are relatively easily displaced by membrane lipids). This helps to explain the experimental observation that cholate is an effective detergent both for solubilizing cytochrome c oxidase and for reconstituting this protein into a defined lipid bilayer environment. An excess of cholate readily displaces almost all of the native phospholipids, and the protein is dispersed in cholate micelles. However, when phospholipids are added back, the cholate molecules at the protein surface are replaced because of the higher relative binding of the phospholipids. Observed differences between the behavior of phosphatidylcholine and phosphatidylglycerol suggest that reconstitution in cholate is a selective process in which detergent molecules in localized areas on the protein surface are more readily displaced by certain phospholipids.     

Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding in solution

Affinity characterization by mass spectrometry (AC–MS) is a novel LC–MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC–MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC–MS was likewise extended to a larger set of soluble protein–ligand systems. It was established as a valuable resource for counter screen and structure–activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.     

Effect of detergents on the association of the glycophorin a transmembrane helix

We have examined the role of the environment on the interactions between transmembrane helices using, as a model system, the dimerization of the glycophorin A transmembrane helix. In this study we have focused on micellar environments and have examined a series of detergents that include a range of alkyl chain lengths, combined with ionic, zwitterionic, and nonionic headgroups. For each we have measured how the apparent equilibrium constant depends on the detergent concentration. In two detergents we also measured the thermal sensitivity of the equilibrium constant, from which we derive the van't Hoff enthalpy and entropy. We show that several simple models are inadequate for explaining our results; however, models that include the effect of detergent concentration on detergent binding are able to account for our measurements. Our analysis suggests that the effects of detergents on helix association are due to a pair of opposing effects: an enthalpic effect, which drives association as the detergent concentration is increased and which is sensitive to the chemical nature of the detergent headgroup, opposed by an entropic effect, which drives peptide dissociation as the detergent concentration is raised. Our results also indicate that the monomer-monomer interface is relatively hydrophilic and that association within detergent micelles is driven by the enthalpy change. The wide variations in glycophorin a dimmer, stability with the detergent used, together with the realization that this results from the balance between two opposing effects, suggests that detergents might be selected that drive association rather than dissociation of peptide dimers.     

Regeneration and duplication following operations in situ on the imaginal discs of Drosophila melanogaster

A technique is described which allows defects to be made in situ in the imaginal discs of immature Drosophila larvae. Bisection of the second leg disc across the upper-lower axis results in regeneration of the remainder of the disc from the upper portion, and mirror-image duplication of the anlagen in the lower half (Figs. 2–5, Table 1). The upper half, retaining its connection to the larval epidermis, is able to evert; the lower half metamorphoses as an uneverted implant. Partial bisection of the disc often results in the production of branched legs in which one branch is complete but the other is a double half (Figs. 7–9). These cases can be interpreted as resulting from regeneration from one cut surface and duplication from the other. Pattern triplications have been obtained by partial bisections of the wing disc (Fig. 10), and these can be interpreted in a similar manner. It is suggested that regeneration and duplication are identical phenomena, resulting from the properties of of the anlagen at the cut edge. Cases of regeneration and duplication in other insect and vertebrate systems are discussed, and interpreted on the basis of gradients of developmental capacity (Fig. 11).     

The immunoglobulin-superfamily molecule basigin is a binding protein for oligomannosidic carbohydrates: an anti-idiotypic approach

Recognition molecules that carry carbohydrate structures regulate cell interactions during development and play important roles in synaptic plasticity and regeneration in the adult. Glycans appear to be involved in these interactions. We have searched for binding proteins for oligomannosidic structures using the L3 antibody directed against high mannose-type glycans in an anti-idiotypic approach. A selected monoclonal anti-idiotype antibody was used for affinity chromatography and identified basigin as a binding protein from mouse brain detergent lysates. Basigin was found to bind to high mannose-carrying cell recognition molecules, such as myelin-associated glycoprotein, L1, the beta2-subunit of Na+/K+-ATPase and an oligomannosidic neoglycolipid. Furthermore, basigin was involved in outgrowth of astrocytic processes in vitro. A striking homology between the first immunoglobulin (Ig)-like domain of basigin and the fourth Ig-like domain of NCAM, previously shown to bind to oligomannosidic glycans, and the lectin domain of the mannose receptor confirms that basigin is an oligomannose binding lectin. To our knowledge this is the first report that anti-idiotypic antibodies can be used to identify binding partners for carbohydrates.     

Isolation of Lamellar Aggregates of the Light-Harvesting Chlorophyll a/b Protein Complex of Photosystem II with Long-Range Chiral Order and Structural Flexibility

Isolation of LHCII, the light-harvesting chlorophyll a/b complex of photosystem II, based on the procedure described by Krupaet al.(1987,Plant Physiol.84, 19–24), was optimized for obtaining purified lamellar aggregates with long-range chiral order and structural flexibility (the capability of undergoing light-induced reversible structural changes). By varying the concentration of the detergent Triton X-100 for the solubilization of thylakoid membranes, we obtained four types of LHCII aggregates: (i) With low detergent concentration, ≤0.6% (v/v), the aggregates contained lipids in high amount. These preparations with Chl a/b ratios of about 1.4 contained minor antenna complexes with a fingerprint of an additional CD band at (+) 505 nm; they formed disordered lamellae and exhibited no or weak psi-type CD bands (psi, polymerization- or salt-induced), which did not possess the ability to undergo light-induced changes (ΔCD). (ii) At the optimal concentration, around 0.7 ± 0.1% (v/v), the detergent removed some lipids and most of the minor complexes, and the Chl a/b ratio dropped to 1.0–1.1. LHCII formed loosely stacked two-dimensional lamellae which exhibited psi-type CD bands and large light-induced reversible structural changes (ΔCD). (iii) At detergent concentration above the optimum, around 0.8–1% (v/v), the lipid content of LHCII decreased and minor complexes could not be detected. LHCII formed disordered aggregates and showed neither psi-type CD nor ΔCD. (iv) High concentrations (≥1.1% (v/v)) Triton X-100 led to very pure but largely delipidated samples assembled into tightly stacked three-dimensional lamellar structures with intense psi-type CD but no ΔCD.     

Monitoring of detergent binding to amphiphilic proteins by means fo micelles containing the hydrophobic dye Sudan Black B

A simple method for detecting micellar binding of Triton X-100 to amphiphilic proteins is described. The hydrophobic dye Sudan Black B is incorporated into Triton micelles. Binding of the coloured micelles to serum apoliproteins, as well as to amphiphilic proteins, of erythrocyte and fat globule membranes renders these visible as dark bands after sucrose density gradient centrifugation. In contrast, the hydrophilic proteins present in lipoprotein-free serum do not show detergent binding. The method does not permit accurate quantification of detergent binding, but may serve as a pilot procedure for initial detection of amphiphilic proteins and for monitoring their isolation from crude solubilized membrane material. The sensitivity of the assay corresponds to that obtained with [³H]Triton X-100.     

On the dissociation and reassociation of MHC class II-foreign peptide complexes. Evidence that brief transit through an acidic compartment is not sufficient for binding site regeneration

The stability of a specific complex between the peptide Ag representing residue 323-339 of OVA and the MHC class II protein, I-Ad, in a lipid bilayer was investigated as a function of pH and temperature. The complex is much more stable in a lipid bilayer than previously reported for detergent micelles. Measureable dissociation was detectable only after several hours at a pH below 5. The results show that a purified preparation of MHC class II molecules can sequentially bind, release, and rebind peptide, indicating that, in principle, MHC class II molecules could be used more than once for peptide binding. However, the time and pH required for peptide-MHC dissociation suggests that, in an Ag presenting cell, either a prolonged residence in an acidic compartment or other factors will be required for regeneration of the peptide binding site.     

Probing the Conformation of FhaC with Small-Angle Neutron Scattering and Molecular Modeling

Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120–160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins.     

Sensory rhodopsin II/transducer complex formation in detergent and in lipid bilayers studied with FRET

The photophobic receptor from Natronomonas pharaonis (NpSRII) forms a photo-signalling complex with its cognate transducer (NpHtrII). In order to elucidate the complex formation in more detail, we have studied the intermolecular binding of both constituents (NpSRII and NpHtrII₁₅₇; truncated at residue 157) in detergent buffers, and in lipid bilayers using FRET. The data for hetero-dimer formation of NpSRII/NpHtrII in detergent agrees well with K_D values (∼ 200 nM) described in the literature. In lipid bilayers, the binding affinity between proteins in the NpSRII/NpHtrII complex is at least one order of magnitude stronger. In detergent the strength of binding is similar for both homo-dimers (NpSRII/NpSRII and NpHtrII/NpHtrII) but significantly weaker (K_D  ∼ 16 μM) when compared to the hetero-dimer. The intermolecular binding is again considerably stronger in lipid bilayers; however, it is not as strong as that observed for the hetero-dimer. At a molar transducer/lipid ratio of 1:2000, which is still well above physiological concentrations, only 40% homo-dimers are formed. Apparently, in cell membranes the formation of the assumed functionally active oligomeric 2:2 complex depends on the full-length transducer including the helical cytoplasmic part, which is thought to tighten the transducer-dimer association.