Search results for "Biophysic"
showing 10 items of 3565 documents
Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain
1999
Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W--K) mutations of avidin and streptavidin were both capable of binding biotin and bioti…
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…
Adsorption and Conformation Behavior of Biotinylated Fibronectin on Streptavidin-Modified TiOX Surfaces Studied by SPR and AFM
2011
It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformati…
Engineering of chicken avidin: a progressive series of reduced charge mutants.
1998
Avidin, a positively charged egg-white glycoprotein, is a widely used tool in biotechnological applications because of its ability to bind biotin strongly. The high pI of avidin (approximately 10.5), however, is a hindrance in certain applications due to non-specific (charge-related) binding. Here we report a construction of a series of avidin charge mutants with pIs ranging from 9.4 to 4.7. Rational design of the avidin mutants was based on known crystallographic data together with comparative sequence alignment of avidin, streptavidin and a set of avidin-related genes which occur in the chicken genome. All charge mutants retained the ability to bind biotin tightly according to optical bio…
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…
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…
Formation of protein multilayers and their competitive replacement based on self-assembled biotinylated phospholipids.
1994
Based on specific recognition processes the build-up of protein multilayers was achieved using streptavidin layers as a docking matrix. For this purpose, streptavidin was organized at biotin-containing monolayers, liposomes, and self-assembled layers on gold. Thus, mixed double and triple layers of streptavidin, Con A, Fab fragments, and hormones were prepared and characterized by fluorescence microscopy and plasmon spectroscopy. Using biotin analogues with lower binding constants several cycles of multilayer formation followed by competitive replacement could be achieved.
Layer-by-Layer Assembly of a Streptavidin–Fibronectin Multilayer on Biotinylated TiOX
2013
The biomodification of surfaces, especially titanium, is an important issue in current biomedical research. Regarding titanium, it is also important to ensure a specific protein modification of its surface because here protein binding that is too random can be observed. Specific nanoscale architectures can be applied to overcome this problem. As recently shown, streptavidin can be used as a coupling agent to immobilize biotinylated fibronectin (bFn) on a TiO(X) surface. Because of the conformation of adsorbed biotinylated fibronectin on a streptavidin monolayer, it is possible to adsorb more streptavidin and biotinylated fibronectin layers. On this basis, an alternating protein multilayer c…
Molecular mechanisms determining the strength of receptor-mediated intermembrane adhesion
1995
The strength of receptor-mediated cell adhesion is directly controlled by the mechanism of cohesive failure between the cell surface and underlying substrate. Unbinding can occur either at the locus of the specific bond or within the bilayer, which results in tearing the hydrophobic anchors from the membrane interior. In this work, the surface force apparatus has been used to investigate the relationship between the receptor-ligand bond affinities and the dominant mechanism of receptor-coupled membrane detachment. The receptors and ligands used in this study were membrane-bound streptavidin and biotin analogs, respectively, with solution affinities ranging over 10 orders of magnitude. With …
Molecular recognition in biotin-streptavidin systems and analogues at the air-water interface
1992
Abstract Specific interaction between biotin and the protein streptavidin in monolayers of synthetic lipids with biotin headgroups has been shown to lead to formation of highly ordered two-dimensional streptavidin crystals. The same behaviour is observed when using desthiobiotin as lipid headgroup which exhibits a significantly lower binding constant compared with biotin (5 × 10 13 M -1 compared with 10 15 M -1 ). This offers the possibility of detaching competetively the 2D crystalline streptavidin layer by addition of free biotin to the aqueous phase. Use of lipoic acid as lipid headgroup ( K a = 7 × 10 7 M −1 ) leads to formation of small snisotropic protein domains indicating a crystall…