Search results for "Streptavidin"
showing 10 items of 54 documents
Specific recognition and formation of two- dimensional streptavidin domains in monolayers: applications to molecular devices
1989
Abstract By virtue of the high-affinity specific interaction between the vitamin, biotin, and the protein, streptavidin, monolayers of synthetic lipids with biotin headgroups can tightly bind streptavidin at the lipid-water interface. Through this specific recognition fluorescently-labelled streptavidin spontaneously organizes in the plane of the interface to form large protein domains, directly visible in situ by fluorescence microscopy and exhibiting optical anisotropy. Further structural characterization has shown that these domains are two-dimensional protein crystals. Correlation with the known three-dimensional crystal structure of streptavidin indicates that two of streptavidin's fou…
Recombinant NeutraLite Avidin: a non-glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific bindin…
2000
AbstractA recombinant non-glycosylated and acidic form of avidin was designed and expressed in soluble form in baculovirus-infected insect cells. The mutations were based on the same principles that guided the design of the chemically and enzymatically modified avidin derivative, known as NeutraLite Avidin. In this novel recombinant avidin derivative, five out of the eight arginine residues were replaced with neutral amino acids, and two of the lysine residues were replaced by glutamic acid. In addition, the carbohydrate-bearing asparagine-17 residue was altered to an isoleucine, according to the known sequences of avidin-related genes. The resultant mutant protein, termed recombinant Neutr…
Surface functionalization and surface recognition: Plasmon optical detection of molecular recognition at self assembled monolayers
1991
The synthesis of biotin- functionalized organic mercaptans and their chemisorption on gold surfaces is described. Biotin bound covalently to self assembled monolayers is recognized by streptavidin from aqueous buffer solutions. Spacer length and packing density of the biotin labels on the organic surface determine the docking kinetics. With a flexible and hydrophilic spacer very fast -diffusion controlled-docking is observed. As an alternative method of self assembly the spreading of organic mercaptans on water surfaces is established. Pressure-area diagrams of different functionalized mercaptans and disulfides are shown and their monolayer properties are discussed.
Interaction between biotin lipids and streptavidin in monolayers: formation of oriented two-dimensional protein domains induced by surface recognitio…
1989
Highly specific ligand-receptor interactions generally characterize surface recognition reactions. Such processes can be simulated by streptavidin-biotin-specific binding. Biotin lipids have thus been synthesized, and their interaction with streptavidin (or avidin) at the air-water interface was directly shown by measurement of surface pressure isotherms and fluorescence microscopy. These proteins interact with the biotin lipid monolayer via specific binding or nonspecific adsorption. Both phenomena were clearly distinguished by use of the inactivated form of streptavidin. The binding of fluorescein-labeled streptavidin to monolayers was also directly observed by fluorescence microscopy. Th…
X-ray diffraction of a protein crystal anchored at the air/water interface
1995
We report the first successful in situ x-ray diffraction experiment with a 2D protein array at the lipid/water interface and demonstrate that the order can be controlled via lateral pressure or density. A protein (streptavidin) was bound to a monolayer of biotinylated lipid at the air/water interface, and diffraction of the protein layer could be measured to many orders. Compression of the monolayer changed the diffraction pattern drastically, indicating that the protein structure can be strongly influenced by external parameters like lateral pressure or density. From the width of the peaks, we find that aggregates consisting of as few as 100 monomers contribute to the diffraction. This ind…
Factors Dictating the Pseudocatalytic Efficiency of Avidins
2006
The hydrolysis of biotinyl p-nitrophenyl ester (BNP) by a series of avidin derivatives was examined. Surprisingly, a hyperthermostable avidin-related protein (AVR4) was shown to display extraordinary yet puzzling hydrolytic activity. In order to evaluate the molecular determinants that contribute to the reaction, the crystal structure of AVR4 was compared with those of avidin, streptavidin and key mutants of the two proteins in complex with biotinyl p-nitroanilide (BNA), the inert amide analogue of BNP. The structures revealed that a critical lysine residue contributes to the hydrolysis of BNP by avidin but has only a minor contribution to the AVR4-mediated reaction. Indeed, the respective …
Protein-membrane interaction probed by single plasmonic nanoparticles.
2008
We present a nanosized and addressable sensor platform based on membrane coated plasmonic particles and show unequivocally the covering with lipid bilayers as well as the subsequent detection of streptavidin binding to biotinylated lipids. The binding is detected on membrane covered gold nanorods by monitoring the spectral shift by fast single particle spectroscopy (fastSPS) on many particles in parallel. Our approach allows for local analysis of protein interaction with biological membranes as a function of the lateral composition of phase separated membranes.
Molecular recognition processes at functionalized lipid surfaces: a neutron reflectivity study
1992
The specific binding of proteins to functionalized monolayers on aqueous subphases has been characterized by neutron reflectivity measurements. As a model for the investigation of a recognition process on a molecular length scale, streptavidin (SA) and biotin were chosen because of the high specific affinity between them. Reflectivities from the aqueous (NaCl/H2O or NaCl/D2O) surfaces covered with the biotin-lipid monolayers before and after the adsorption of proteins were collected with a novel, fixed wavelength liquid surface neutron reflectometer. In quantitative terms, binding was found to occur at a biotin surface concentration as low as 1 molecule/1250 A2 (compare to ∼ 1 molecule/40 A…
Oligodendrocytes in mouse corpus callosum are coupled via gap junction channels formed by connexin47 and connexin32.
2010
According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole-cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction-permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin-Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GF…
Specific interaction of desthiobiotin lipids and water-soluble biotin compounds with streptavidin
1991
As shown for biotin lipids (Ref. 1), the formation of perfect 2-D crystalline streptavidin domains can also be observed in the plane of desthiobiotin lipid monolayers. The binding constant of streptavidin with desthiobiotin (Ka = 5·1013 mol−1) is lower than that with biotin (Ka = 1015 mol−1) (Ref. 2). By adding free biotin into the subphase a competitive replacement and a detaching of the streptavidin domains from the desthiobiotin lipid monolayer takes place. Streptavidin domains built at receptor lipid monolayers are still functional. As could be shown, there are two biotin binding sites at each protein molecule that are fully accessible to biotin (Ref. 1). This can be proven by the inter…