Search results for "avidin"
showing 10 items of 97 documents
Construction of Chimeric Dual-Chain Avidin by Tandem Fusion of the Related Avidins
2011
BackgroundAvidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences i…
DNA origami as a nanoscale template for protein assembly
2009
We describe two general approaches to the utilization of DNA origami structures for the assembly of materials. In one approach, DNA origami is used as a prefabricated template for subsequent assembly of materials. In the other, materials are assembled simultaneously with the DNA origami, i.e. the DNA origami technique is used to drive the assembly of materials. Fabrication of complex protein structures is demonstrated by these two approaches. The latter approach has the potential to be extended to the assembly of multiple materials with single attachment chemistry.
A DNA-nanoparticle actuator enabling optical monitoring of nanoscale movements induced by an electric field.
2018
Merging biological and non-biological matter to fabricate nanoscale assemblies with controllable motion and function is of great interest due to its potential application, for example, in diagnostics and biosensing. Here, we have constructed a DNA-based bionanoactuator that interfaces with biological and non-biological matter via an electric field in a reversibly controllable fashion. The read-out of the actuator is based on motion-induced changes in the plasmon resonance of a gold nanoparticle immobilized to a gold surface by single stranded DNA. The motion of the gold nanoparticle and thus the conformational changes of the DNA under varying electric field were analyzed by dark field spect…
Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol
1997
Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, h…
Design and construction of highly stable, protease-resistant chimeric avidins.
2005
The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and …
Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding
2003
AbstractIn order to turn the subunit association and biotin binding of avidin into pH-sensitive phenomena, we have replaced individually three amino acid residues in avidin (Met96, Val115 and Ile117) with histidines in the 1–3 interface, and in combination with a histidine conversion in the 1–2 interface (Trp110). The single replacements Met96His and Val115His in the 1–3 interface were found to have a clear effect on the quaternary structure of avidin, since subunit associations of these mutants became pH-dependent. The histidine replacement in the 1–2 interface affected the biotin-binding properties of the mutants, in particular reversibility of binding and protein–ligand complex formation…
Construction of a dual chain pseudotetrameric chicken avidin by combining two circularly permuted avidins.
2004
Two distinct circularly permuted forms of chicken avidin were designed with the aim of constructing a fusion avidin containing two biotin-binding sites in one polypeptide. The old N and C termini of wild-type avidin were connected to each other via a glycine/serine-rich linker, and the new termini were introduced into two different loops. This enabled the creation of the desired fusion construct using a short linker peptide between the two different circularly permuted subunits. The circularly permuted avidins (circularly permuted avidin 5 → 4 and circularly permuted avidin 6 → 5) and their fusion, pseudotetrameric dual chain avidin, were biologically active, i.e. showed biotin binding, and…
Tetravalent single-chain avidin: from subunits to protein domains via circularly permuted avidins
2005
scAvd (single-chain avidin, where two dcAvd are joined in a single polypeptide chain), having four biotin-binding domains, was constructed by fusion of topologically modified avidin units. scAvd showed similar biotin binding and thermal stability properties as chicken avidin. The DNA construct encoding scAvd contains four circularly permuted avidin domains, plus short linkers connecting the four domains into a single polypeptide chain. In contrast with wild-type avidin, which contains four identical avidin monomers, scAvd enables each one of the four avidin domains to be independently modified by protein engineering. Therefore the scAvd scaffold can be used to construct spatially and stoich…
Binding Properties of HABA-Type Azo Derivatives to Avidin and Avidin-Related Protein 4
2006
Summary The chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to D -biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands β3 and β4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 A) X-ray stru…
Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin.
2006
Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudotetrameric quaternary assemblies because of symmetry at the monomer-monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilis…