Israel Pecht

Proteins as Electronic Materials: Electron Transport through Solid-State Protein Monolayer Junctions

Electron transfer (ET) through proteins, a fundamental element of many biochemical reactions, is studied intensively in aqueous solutions. Over the past decade, attempts were made to integrate proteins into solid-state junctions in order to study their electronic conductance properties. Most such studies to date were conducted with one or very few molecules in the junction, using scanning probe techniques. Here we present the high-yield, reproducible preparation of large-area monolayer junctions, assembled on a Si platform, of proteins of three different families: azurin (Az), a blue-copper ET protein, bacteriorhodopsin (bR), a membrane protein-chromophore complex with a proton pumping function, and bovine serum albumin (BSA). We achieve highly reproducible electrical current measurements with these three types of monolayers using appropriate top electrodes. Notably, the current-voltage (I-V) measurements on such junctions show relatively minor differences between Az and bR, even though the latter lacks any known ET function. Electron Transport (ETp) across both Az and bR is much more efficient than across BSA, but even for the latter the measured currents are higher than those through a monolayer of organic, C18 alkyl chains that is about half as wide, therefore suggesting transport mechanism(s) different from the often considered coherent mechanism. Our results show that the employed proteins maintain their conformation under these conditions. The relatively efficient ETp through these proteins opens up possibilities for using such biomolecules as current-carrying elements in solid-state electronic devices.

Immunoreceptor tyrosine-based inhibition motif-bearing receptors regulate the immunoreceptor tyrosine-based activation motif-induced activation of immune competent cells

ITIM-bearing receptors, a family which only recently has been recognized, play a key role in the regulation of the ITAM-induced activation of immune competent cells. The mechanism of ITM-mediated regulation in various cells was recently clarified. The present review focuses on ITIM bearing membrane proteins that negatively regulate the activation of cells when co-crosslinked with ITAM containing receptors, illustrates the inhibitory processes by the negative regulation of B-, NK-, T-cells and mast cells and summarizes current views on the mechanism of ITIM-mediated inhibition.

Regulation of the mast cell response to the type 1 Fcε receptor

Summary:  The type I Fcε receptor (FcεRI) is one of the better understood members of its class and is central to the immunological activation of mast cells and basophils, the key players in immunoglobulin E (IgE)‐dependent immediate hypersensitivity. This review provides background information on several distinct regulatory mechanisms controlling this receptor’s stimulus–response coupling network. First, we review the current understanding of this network’s operation, and then we focus on the inhibitory regulatory mechanisms. In particular, we discuss the different known cytosolic molecules (e.g. kinases, phosphatases, and adapters) as well as cell membrane proteins involved in negatively regulating the FcεRI‐induced secretory responses. Knowledge of this field is developing at a fast rate, as new proteins endowed with regulatory functions are still being discovered. Our understanding of the complex networks by which these proteins exert regulation is limited. Although the scope of this review does not include addressing several important biochemical and biophysical aspects of the regulatory mechanisms, it does provide general insights into a central field in immunology.

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl‐ channels in rat mucosal‐type mast cells (line RBL‐2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl‐ ion channels as detected by the patch‐clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and −100 mV, and an increasing open‐state probability with increasing depolarization. Activation of apparently the same Cl‐ channels could be mimicked without stimulation by isolating inside‐out membrane patches in tyrode solution. Parallel inhibition of both Cl‐ channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl‐ channel blocker 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) and the anti‐allergic drug cromolyn. NPPB inhibited both the antigen‐induced Cl‐ current and the serotonin release, where half‐maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl‐ channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside‐out membrane patch. The observed Cl‐ channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl‐ channel in mediator secretion from the mast cells studied.

Electronic Transport via Proteins

A central vision in molecular electronics is the creation of devices with functional molecular components that may provide unique properties. Proteins are attractive candidates for this purpose, as they have specific physical (optical, electrical) and chemical (selective binding, self-assembly) functions and offer a myriad of possibilities for (bio-)chemical modification. This Progress Report focuses on proteins as potential building components for future bioelectronic devices as they are quite efficient electronic conductors, compared with saturated organic molecules. The report addresses several questions: how general is this behavior; how does protein conduction compare with that of saturated and conjugated molecules; and what mechanisms enable efficient conduction across these large molecules? To answer these questions results of nanometer-scale and macroscopic electronic transport measurements across a range of organic molecules and proteins are compiled and analyzed, from single/few molecules to large molecular ensembles, and the influence of measurement methods on the results is considered. Generalizing, it is found that proteins conduct better than saturated molecules, and somewhat poorer than conjugated molecules. Significantly, the presence of cofactors (redox-active or conjugated) in the protein enhances their conduction, but without an obvious advantage for natural electron transfer proteins. Most likely, the conduction mechanisms are hopping (at higher temperatures) and tunneling (below ca. 150-200 K).

SH2 Domain-Containing Inositol Polyphosphate 5′-Phosphatase Is the Main Mediator of the Inhibitory Action of the Mast Cell Function-Associated Antigen

The mast cell function-associated Ag (MAFA) is a type II membrane glycoprotein originally found on the plasma membrane of rat mucosal-type mast cells (RBL-2H3 line). A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering has previously been shown to suppress the secretory response of these cells to the FcεRI stimulus. Here we show that the tyrosine of the ITIM undergoes phosphorylation, on MAFA clustering, that is markedly enhanced on pervanadate treatment of the cells. Furthermore, the Src homology 3 domain of the protein tyrosine kinase Lyn binds directly to a peptide containing nonphosphorylated MAFA ITIM and PAAP motif. Results of both in vitro and in vivo experiments suggest that Lyn is probably responsible for this ITIM phosphorylation, which increases the Src homology domain 2 (SH2) affinity of Lyn for the peptide. In vitro measurements established that tyrosine-phosphorylated MAFA ITIM peptides also bind the SH2 domains of inositol 5′-phosphatase (SHIP) as well as protein tyrosine phosphatase-2. However, the former single domain is bound 8-fold stronger than both of the latter. Further support for the role of SHIP in the action of MAFA stems from in vivo experiments in which tyrosine-phosphorylated MAFA was found to bind primarily SHIP. In RBL-2H3 cells overexpressing wild-type SHIP, MAFA clustering causes markedly stronger inhibition of the secretory response than in control cells expressing normal SHIP levels or cells overexpressing either wild-type protein tyrosine phosphatase-2 or its dominant negative form. In contrast, on overexpression of the SH2 domain of SHIP, the inhibitory action of MAFA is essentially abolished. Taken together, these results suggest that SHIP is the primary enzyme responsible for mediating the inhibition by MAFA of RBL-2H3 cell response to the FcεRI stimulus.

Dynamics of hapten-antibody interaction. Studies on a myeloma protein with anti-2, 4-dinitrophenyl specificity

Abstract The kinetics of interaction between IgA immunoglobulin produced by the mouse plasma cell tumor MOPC 315 and its ligands have been investigated by the temperature-jump-chemical relaxation method. This protein was previously shown to have homogeneous binding sites with affinity towards dinitrophenyl and trinitrophenyl derivatives, comparable to conventionally induced antibodies. The homogeneity of the protein 315 allows a precise analysis of antibody-hapten reaction dynamics, which hitherto were investigated on heterogeneous antibody populations. Three different preparations of the 315 protein were studied for their interaction with either ϵ, N -DNP † -lysine or DNP-glycine: (1) polydisperse form obtained from the serum; (2) monomeric form obtained on mild reduction and alkylation; (3) Fab′ fragment obtained after cleavage with pepsin. In all three cases only a single relaxation time was observed over a broad concentration range. From the dependence of the relaxation times on the free protein and ligand concentrations a single-step binding mechanism was deduced and the specific rates for binding and dissociation were calculated. For all three protein forms, very similar rate constants were observed, implying that there is no effect on the binding dynamics of the protein by either the Fc portion of the molecule or the state of aggregation.

Kinetics of Antibody-Hapten Interactions

The immune system, which is the major defence complex present in vertebrates against foreign cells and pathogens, is comprised of lymphocytes and their products, the antibodies. The latter carry out the function of recognition of antigenic determinants and the resultant triggering of a wide range of biological responses. Antibodies are all immunoglobulins - a group of multichain proteins whose similar gross structure may be expressed as (HL)2n where H and L are the heavy and light polypeptide chains, respectively, linked together by noncovalent and disulfide bonds. For the IgG class n = 1 and,for the IgM class n = 5. The light chains are 22,500 daltons whereas the heavy chainsary according to class between 53,000 for the IgG to 75,000 daltons for IgE (EDELMAN and GALL, 1969). A typical property of antibodies, even from one individual animal and having the same specificity, is the pronounced heterogeneity found in their primary structure. This heterogeneity is confined, however, to the first 110 N-terminal residues of the two chains (variable region) and expresses the diversity of the antigen combining site. Certain stretches in the variable region were found to exhibit higher variability, and these hypervariable residues were proposed to form the main contact areas of the binding site (RABAT and WU, 1971). X-ray crystallography has verified this hypothesis (DAVIES et al., 1976; POLJAK, 1975).

The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase

The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process is induced following reduction of the type 1 Cu(II) by radicals produced by pulse radiolysis. The reversible ET reaction proceeds with a rate constant k ET=k 1→2+k 2→1 of 450±30 s−1 at pH 7.0 and 298 K. The equilibrium constant K was determined to be 0.7 at 298 K from which the individual rate constants for the forward and backward reactions were calculated to be: k 1→2=185±12 s−1 and k 2→1265±18 s−1. The temperature dependence of K allowed us to determine the ΔH° value of the ET equilibrium to be 12.1 kJ mol−1. Measurements of the temperature dependence of the ET process yielded the following activation parameters: forward reaction, ΔH ≠=22.7±3.4 kJ mol−1 and ΔS ≠=−126±11 J K−1 mol−1; backward reaction, ΔH ≠=10.6±1.7 kJ mol−1 and ΔS ≠=−164±15 J K−1 mol−1. X‐ray crystallographic studies of NiRs suggest that the most probable ET pathway linking the two copper sites consists of Cys136, which provides the thiolate ligand to the type 1 copper ion, and the adjacent His135 residue with its imidazole being one of the ligands to the type 2 Cu ion. This pathway is essentially identical to that operating between the type 1 Cu(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared. The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate oxidase.

A new member of the C-type lectin family is a modulator of the mast cell secretory response.

A glycoprotein identified on RBL-2H3 cells as capable of inhibiting the secretory response induced by the type I Fc epsilon receptor was named mast-cell-function-associated antigen (MAFA). The amino acid sequence deduced from the cloned full-length cDNA has now shown that the MAFA has marked sequence homology with several members of the C-type (calcium-dependent) animal lectin family. The high conservation of cysteinyl residues suggests an important role for intrachain disulfide bonds in attaining its structure and biological activity. We further show that MAFA clustering by monoclonal antibody G63 also inhibits the de novo synthesis and secretion of interleukin-6 induced by the Fc epsilon RI stimulus. Though no ligand has yet been identified for the MAFA, experiments using antisense oligonucleotides suggest that this novel lectin may have a role in cell adhesion in addition to its immunomodulatory capacity.