Biotin interaction with

Fig. 2 Secondary, tertiary, and quantenary structures of streptavidin. a Amino acid residues interacting with biotin, b Anti-parallel p sheets with dotted lines showing hydrogen bonds, c Folded apostreptavidin subunit with an extended hairpin loop near the carboxyl terminus, d Formation of streptavidin dimer through the interaction between the extended hairpin loops of the monomeric subunits, e Formation of streptavidin tetramer through the weak interaction of two stable dimer subunits (biotin is shown in pink) N N-terminus, C C-terminus. (a taken from [14]. b taken from [15]. e taken from http //www.scrippslabs.com/graphics/pdfs/Strept.pdf)...

Other proteins that interact with biotin including egg yolk biotinbinding proteins and biotin transport components from several systems including yeast, mammalian intestinal cells, and bacteria [70-73]. Note that although there are other proteins (e.g., fibropellins) that have a motif [DENY]-jc(2)-[KRI]-[STA]-x(2)-V-G-jc-[DN]-jc[FW]-T-[KR] in common with strept(avidin), most if not all of these protein do not bind biotin (http //www.expasy.org/prosite/). 

Each ACC half-reaction is catalyzed by a different protein sub-complex. The vitamin biotin is covalently coupled through an amide bond to a lysine residue on biotin carboxyl carrier protein (BCCP, a homodimer of 16.7-kDa monomers encoded by accB) by a specific enzyme, biotin-apoprotein ligase (encoded by birA), and is essential to activity. The crystal and solution structures of the biotinyl domain of BCCP have been determined, and reveal a unique thumb required for activity (J. Cronan, 2001). Carboxylation of biotin is catalyzed by biotin carboxylase (encoded by accC), a homodimeric enzyme composed of 55-kDa subunits that is copurified complexed with BCCP. The accB and accC genes form an operon. The three-dimensional structure of the biotin carboxylase subunit has been solved by X-ray diffraction revealing an ATP-grasp motif for nucleotide binding. The mechanism of biotin carboxylation involves the reaction of ATP and CO2 to form the shortlived carboxyphosphate, which then interacts with biotin on BCCP for CO2 transfer to the I -nitrogen. 

Fig. 6-9 Target identification of a suppressor of rapamycin [42]. (a) SMIR4 a suppressor of rapamycin identified using a chemical-genetic modifier screen, (b) Identification of gene products that interact with biotin-SMIR4 using a yeast protein microarray [42].

Native ovoflavoprotein (49 kDa, pf=5.1) has, as does ovomucoid, certain antinutritional effects, as it inhibits serine proteases (trypsin, chymotrypsin and also microbial proteases) and has antiviral activity. Ovomacroglobulin (ovostatin) is an inhibitor of serine, cysteine, thiol and metalloproteases and shows antimicrobial activity. Some antinutritional effects are also seen in the basic glycoprotein avidin in raw egg white (relative molecular weight of the monomer is 15.6 kDa). It contains four identical subunits (pf = 9.5), each of which binds one molecule of biotin to give an unavailable complex. However, the denatured avidin, for example in hard-boiled eggs, does not interact with biotin. The interaction of riboflavin with flavoprotein (32 kDa, pf = 4.0) has, on the contrary, a positive influence on vitamin stability. Cystatin acts as cysteine protease inhibitor, and shows antimicrobial, antitumor and immunomodulating activities. 

In a recent example, Kim et al. showed how the combination of several types of nanoparticles could also be wisely used for the design of signaling protocols. They designed a FRET-based inhibition assay to determine the avidin concentration in solution with AuNPs and QDs. The ensemble involves the use of streptavidin-conjugated QDs that interact with biotin-AuNPs through well-known streptavidin-biotin chemistry. This system was not luminescent due to FRET interaction between QDs and AuNPs. Addition of avidin to this ensemble caused the luminescence to increase gradually because the AuNPs were displaced from the streptavidin-functionalized QDs as a consequence of avidin-biotin interactions (Fig. 8). 

Lee G U, Kidwell D A and Colton R J 1994 Sensing discrete streptavidin-biotin interactions with atomic force microscopy Langmuir 10 354... 

Figure Bl.20.10. Typical force curve for a streptavidin surface interacting with a biotin surface in an aqueous electrolyte of controlled pH. This result demonstrates the power of specific protein interactions. Reproduced with pennission from [81].

S. Miyamoto and P. A. Kollman. Absolute and relative binding free energy calculations of the interaction of biotin and its analogs with streptavidin using molecular dynamics/free energy perturbation approaches. Proteins, 16 226-245, 1993. 

Miyamoto S and P A Kollman 1993a. Absolute and Relative Binding Tree Energy Calculations of the Interaction of Biotin and its Analogues with Streptavidin Using Molecular Dynamics/Free Energy Perturbation Approaches. Proteins Structure, Function and Genetics 16 226-245. 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

Figure 6.3 Mts-Atf-Biotin can be used to label bait proteins at available thiol groups using the MTS group, which forms a disulfide linkage after reaction. The modified protein then is allowed to interact with a protein sample and photoactivated with UV light to cause a covalent crosslink with any interacting proteins. Cleavage of the disulfide bond effectively transfers the biotin label to the unknown interacting protein.

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

Using a similar approach, Clq has been modified with biotin-HPDP and allowed to interact with its specific receptor. Subsequent purification of the Clq receptor was accomplished through cleavage of the disulfide bridge of the biotinylation reagent (Ghebrehiwet et al., 1988). 

In some cases, the ability to modify glycans at the reducing end without reduction preserves the carbohydrate s native structure sufficiently to allow interactions with proteins that would otherwise not interact if the bond were reduced. Therefore, depending on the ultimate use of the biotinylated carbohydrate, using a hydrazide mediated conjugation process can have advantages over the use of amine-biotin compounds. 

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