Design of artificial metalloenzymes
20
Citation
34
Reference
10
Related Paper
Citation Trend
Abstract:
Homogeneous and enzymatic catalysis offer complementary means to generate enantiomerically pure compounds. For this reason, in a biomimetic spirit, efforts are currently under way in different groups to design artificial enzymes. Two complementary strategies are possible to incorporate active organometallic catalyst precursors into a protein environment. The first strategy utilizes covalent anchoring of the organometallic complexes into the protein environment. The second strategy relies on the use of non-covalent incorporation of the organometallic precursor into the protein. In this review, attention is focused on the use of semisynthetic enzymes to produce efficient enantioselective hybrid catalysts for a given reaction. This article also includes our recent research results and implications in developing the biotin–avidin technology to localize the biotinylated organometallic catalyst precursor within a well-defined protein environment. Copyright © 2005 John Wiley & Sons, Ltd.Keywords:
Avidin
Enzyme Catalysis
Cite
Avidin
Cite
Citations (100)
Initial contact between the opportunistic fungal pathogen Candida albicans and host tissue occurs at the cell surface. Biotin derivatives have been used to label the cell-surface proteins of yeasts, with labelled proteins subsequently detected by avidin–reporter conjugates. Previous work has indicated that avidin can bind to C. albicans proteins in the absence of biotin, suggesting a possible host-cell-recognition mechanism by fungal cell-surface proteins. To investigate this mechanism, Western blots of proteins extracted from biotinylated and mock-treated cells were probed with avidin or modified-avidin reagents. Each avidin reagent bound to cell-wall proteins extracted from non-biotinylated cells. Binding did not appear to be due to the lectin-like activity of the cell-wall proteins of C. albicans or to the presence of biotin in the sample itself. Binding was inhibited by added biotin, by the chaotrope KSCN and by NaCl in a concentration-dependent manner, although inhibition varied among the avidin conjugates tested. Thus, the non-specific binding of avidin to the cell-wall proteins of C. albicans appears to involve hydrophobic and electrostatic interactions, depending on the particular avidin species. These observations demonstrate potential pitfalls in the use of avidin–biotin complexes to identify cell-surface molecules and could provide insights into protein–protein interactions at the C. albicans cell wall.
Avidin
Cite
Citations (19)
Biotinylated bait molecules can be immobilized on biotinylated sensor chips by formation of biotin–avidin–biotin bridges which are very stable when using wild-type (strept)avidin. Stable immobilization of biotinylated baits is important for monitoring reversible binding and dissociation of prey molecules. For measurements with another bait molecule, however, it is desirable to replace all immobilized proteins by fresh (strept)avidin and new biotinylated bait. In this study, five avidin mutants have been characterized with respect to their ability to form switchable biotin–avidin–biotin bridges on biotinylated chip surfaces, as needed for complete chip regeneration. All five mutants formed stable biotin–avidin–biotin bridges at pH 7, were more or less stable at pH 2–3, and required the combination of pH 2 with SDS for quantitative removal from the chip surface. Mutant #3 ("switchavidin") showed the best combination of properties, i.e., low nonspecific adsorption of protein and nucleic acids, high binding capacity, and good stability at pH 2–3, as typically used for quantitative removal of prey molecules in repeated measurement cycles.
Avidin
Streptavidin
Cite
Citations (11)
Avidin
Streptavidin
Cite
Citations (19)
We report the synthesis, characterization, and avidin-binding properties of two novel ruthenium complexes, [Ru(bpy)2(phen-biotin)][PF6]2 1 and [Ru(phen)2(phen-biotin)][PF6]2 2 (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline, phen-biotin = 5-(10-amidobiotinyl)-1,10-phenanthroline)). We demonstrate that both biotinylated compounds bind to avidin through their biotin moieties with high affinity and in a 4:1 ratio. The binding of compounds 1 and 2 to avidin results in an enhancement in luminescence intensity (∼1.4×, ∼1.6×, respectively), relative to the unbound biotinylated ruthenium complexes. This behavior is markedly different from biotinylated organic dyes, whose fluorescence is quenched upon binding to avidin. Thus, ruthenium−biotin complexes 1 and 2 can form the basis of new, simplified biotin−avidin assays, which involve luminescence detection of the relevant biotinylated molecule through cross-linking with avidin.
Avidin
Phenanthroline
Bipyridine
Cite
Citations (28)
Avidin
Sepharose
Cite
Citations (21)
Avidin
Cite
Citations (0)
Avidin
Cite
Citations (30)
Switchavidin is a chicken avidin mutant displaying reversible binding to biotin, an improved binding affinity toward conjugated biotin, and low nonspecific binding due to reduced surface charge. These properties make switchavidin an optimal tool in biosensor applications for the reversible immobilization of biotinylated proteins on biotinylated sensor surfaces. Furthermore, switchavidin opens novel possibilities for patterning, purification, and labeling.
Avidin
Streptavidin
Cite
Citations (33)
Avidin
Conjugate
Biomolecule
Streptavidin
Cite
Citations (42)