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PubMed1990

Biotinyl-high-density lipoproteins as a probe for the determination of high-density lipoprotein turnover in humans.

A simple and reliable method has been developed for the determination of high-density lipoprotein (HDL) turnover in humans. In this method, complex formation of [14C]methylavidin with biotinyl-HDL3 and subsequent precipitation of excess [14C]methylavidin with biotinyl-silica-gel is utilized for the detection of as little as 0.1 microgram of biotinyl-HDL3 (by protein)/ml of serum with high precision and reproducibility, at 7.9 +/- 0.9% (n = 7) of the peptidyl lysine modified. In serial dilutions and quadruplicate determinations the intra- and interassay variations were less than 4.7% and 5.2%, respectively (n = 5). Recovery of biotinyl-HDL3 averaged 92 +/- 5.7% throughout the working range of the assay (n = 4). Variations in the levels of HDL or very-low-density lipoprotein (VLDL) did not interfere with the measurement of biotinyl-HDL3 in serum. Also, results were not affected by storage of serum samples at -80 degrees C for up to 4 weeks. After reinjection of autologous biotinyl-HDL3 (0.12 mg protein/kg body wt.) into five normolipidemic male volunteers, typical decay curves were obtained. The mean half-life of biotinyl-HDL3 was 5.1 +/- 0.5 days, no different from that reported for radiolabeled HDL or radiolabeled HDL apolipoproteins. Routine immunobinding analysis did not reveal formation of antibodies with specificity towards HDL 4 weeks after the reinjection studies. From this it appears that biotinyl-HDL3 is a suitable probe and a safe and reliable alternative for the determination of HDL turnover in humans when application of radiolabeled HDL is not desirable.

Adult↗
PubMed1995

Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing.

BACKGROUND: Acetyl-coenzyme A carboxylase catalyzes the first committed step of fatty acid biosynthesis. Universally, this reaction involves three functional components all related to a carboxybiotinyl intermediate. A biotinyl domain shuttles its covalently attached biotin prosthetic group between the active sites of a biotin carboxylase and a carboxyl transferase. In Escherichia coli, the three components reside in separate subunits: a biotinyl domain is the functional portion of one of these, biotin carboxy carrier protein (BCCP). RESULTS: We have expressed natural and selenomethionyl (Se-met) BCCP from E. coli as biotinylated recombinant proteins, proteolyzed them with subtilisin Carlsberg to produce the biotinyl domains BCCP and Se-met BCCPsc, determined the crystal structure of Se-met BCCPsc using a modified version of the multiwavelength anomalous diffraction (MAD) phasing protocol, and refined the structure for the natural BCCPsc at 1.8 A resolution. The structure may be described as a capped beta sandwich with quasi-dyad symmetry. Each half contains a characteristic hammerhead motif. The biotinylated lysin is located at a hairpin beta turn which connects the two symmetric halves of the molecule, and its biotinyl group interacts with a non-symmetric protrusion from the core. CONCLUSIONS: This first crystal structure of a biotinyl domain helps to unravel the central role of such domains in reactions catalyzed by biotin-dependent carboxylases. The hammerhead structure observed twice in BCCPsc may be regarded as the basic structural motif of biotinyl and lipoyl domains of a superfamily of enzymes. The new MAD phasing techniques developed in the course of determining this structure enhance the power of the MAD method.

Acetyl-CoA Carboxylase↗
PubMed1975

Isolation of peptides from the carboxyl carrier subunit of transcarboxylase. Role of the non-biotinyl peptide in assembly.

Transcarboxylase is made up of a central hexameric subunit (S20,W similar 12 S), three peripheral dimeric metallo subunits (S20,W similar to 5 S), and six biotinyl carboxyl carrier subunits (S20,W similar to 1.3 S). The results presented here show that the carboxyl carrier subunit is required for assembly of the 12S and 5S subunits into the oligomer. However, only a portion of the subunit is required for assembly. On treatment of transcarboxylase briefly with trypsin at pH 6.3 extremely susceptible peptide bonds of the carboxyl carrier protein are cleaved releasing biotinyl peptides of about similar to 66 and similar to 40 residues. The resulting trypsinized transcarboxylase, though enzymatically inactive, remains essentially intact as judged by its hydrodynamic and molecular sieving properties. The modified enzyme can be dissociated at pH 8 to the central 12S subunit and peripheral 5S subunit to which the residual portion(s) of the cleaved carboxyl carrier protein is still attached. These components can then be separated by molecular sieving. The residual portion of the carboxyl carrier protein (non-biotinyl peptide) can then be isolated by dissociation of the 5S subunit complex at pH 9 and by chromatography over Bio-Gel A-1.5m. The isolated non-biotinyl peptide has been shown to contain the combining domain of the 1.3SE carboxyl carrier protein since it causes combination of the 12S and 5S subunits. Active enzyme is formed by combination of the intact carboxyl carrier protein and the 12S and 5S subunits and an inactive oligomer of similar size is formed if the non-biotinyl peptide is used in place of the carboxyl carrier protein. The similar to 66- and similar to 40-residue biotinyl peptides, which are released by the trypsin treatment, apparently occur on an exposed portion of the enzyme. This portion of the carboxyl carrier protein apparently serves to place the biotinyl group adjacent to the two substrate sites of the enzyme, one of which is on the peripheral subunit and the other on the central subunit. Thus the carboxyl carrier protein has two functions: one portion holds the 12S and 5S subunits in juxtaposition and the other portion orients the biotinyl group adjacent to the substrate sites so that it may function as a carboxyl carrier between the sites.

Acetyltransferases↗
PubMed1999

Solution structures of apo and holo biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy.

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP. In agreement with X-ray crystallographic studies [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues. The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101. However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.

Acetyl-CoA Carboxylase↗
PubMed1985

A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin.

Purified bovine brain calmodulin was biotinylated with biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide. Biotinylated calmodulin was used to detect and quantify calmodulin-binding proteins following both protein blotting and slot-blot procedures by using alkaline phosphatase or peroxidase coupled to avidin. When purified bovine brain calcineurin, a calmodulin-dependent protein phosphatase, was immobilized on nitrocellulose slot blots, biotinylated calmodulin bound in a calcium-dependent saturable manner; these blots were then quantified by densitometry. Biotinylated calmodulin was able to detect as little as 10 ng of calcineurin, and the binding was competitively inhibited by addition of either native calmodulin or trifluoperazine. When biotinylated calmodulin was used to probe protein blots of crude brain cytosol and membrane preparations after gel electrophoresis, only protein bands characteristic of known calmodulin-binding proteins (i.e., calmodulin-dependent protein kinase, calcineurin, spectrin) were detected with avidin-peroxidase or avidin-alkaline phosphatase procedures. Purified calcineurin was subjected to one- and two-dimensional gel electrophoresis and protein blotting; as expected, only the 61-kDa calmodulin-binding subunit was detected. When the two-dimensional protein blot was incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase, several apparent forms of the 61-kDa catalytic subunit were detected, consistent with isozymic species of the enzyme. The results of these studies suggest that biotinylated calmodulin can be used as a simple, sensitive, and quantifiable probe for the study of calmodulin-binding proteins.

Animals↗
PubMed1992

Fast lysis by complement and uptake by liver of avidin-carrying biotinylated erythrocytes.

The fate of 51Cr-labelled avidin-carrying biotinylated erythrocytes after intravenous injection in the rat was examined. Surface amino groups of the erythrocyte membrane were modified by biotin N-hydroxysuccinimide ester. The biodistribution and stability of biotinylated erythrocytes in the blood were similar to those of non-biotinylated cells. Both types of cells circulated in the bloodstream for prolonged periods of time without substantial lysis (about 2-3% of injected radioactivity per g of blood for 24-48 hours, no more than 2% of lysis). Both types of erythrocytes were cleared by the spleen. The clearance of biotinylated cells was faster and more pronounced (peak of spleen uptake at 3 hours after injection, up to 35% of injected radioactivity per g of spleen), than that of nonbiotinylated cells (peak of spleen uptake at 24 hours after injection, up to 25% of injected radioactivity per g of spleen). Attachment of avidin to biotinylated cells results in extremely rapid lysis and clearance from the bloodstream (0.17% of injected radioactivity per g of blood 30 min after injection, 100% lysis). Radioactivity was rapidly cleared by the liver (up to 80% of injected dose per g of tissue, 70% per organ). Uptake by the spleen plays only a minor role in the clearance. Considerable lung uptake of avidin-carrying biotinylated erythrocytes was observed. Avidin-carrying biotinylated erythrocytes were lysed by fresh homologous serum in vitro in contrast to biotinylated and native cells. Lysis was eliminated by pretreatment of serum with EDTA or heating, which indicates a complement-dependent mechanism of lysis.

Animals↗
PubMed1987

Biotinylation of low density lipoproteins via free amino groups without loss of receptor binding activity.

Low density lipoproteins were biotinylated via free amino groups using carbodiimide-activated biotin or D-biotin-N-hydroxysuccinimide ester. The receptor binding activity of the biotinylated LDL was determined by their ability to displace 125I-labeled LDL from the rat hepatic LDL receptor in the liposome filtration assay. LDL biotinylated with either of the two reagents was able to compete effectively with 125I-labeled LDL providing less than twenty biotin moieties were incorporated per lipoprotein particle. When more than twenty biotins were linked there was a marked loss of activity. The following conditions were adopted as standard for the biotinylation of LDL via free amino groups: 0.3 mumol of D-biotin-N-hydroxysuccinimide ester was incubated with 2 mg of LDL for 1 hr at room temperature. These conditions reproducibly yielded 11.3 +/- 0.6 biotins per LDL particle. With the biotinylated LDL and a performed streptavidin-biotinylated peroxidase complex, the hepatic LDL receptor from rats treated with 17 alpha-ethinyl estradiol was visualized as a single band on electroblots. Finally, the biotinylated LDL was used in an enzyme-linked sorbent assay for the LDL receptor. When solubilized liver membrane proteins from rats treated with 17 alpha-ethinyl estradiol were fixed to the wells with 1.6% paraformaldehyde, a specific binding greater than 0.4 absorbance units was observed which was about ninefold higher than with solubilized proteins from normal rats. We therefore suggest that D-biotin-N-hydroxysuccinimide ester can be used to biotinylate LDL reliably without destroying the lipoprotein's ability to bind specifically to its high affinity receptor.

Animals↗
PubMed1998

Improving tumour targeting and decreasing normal tissue uptake by optimizing the stoichiometry of a two-step biotinylated-antibody/streptavidin-based targeting strategy: studies in a nude mouse xenograft model.

We aimed to study in detail the in vivo stoichiometry of the individual elements of the 2-step streptavidin based approach to tumour targeting, in a nude mouse xenograft model, by the administration of a first step consisting of biotinylated anti-tumour specific antibody and a second step consisting of streptavidin. This process was undertaken to identify the optimum conditions for radiotherapeutic tumour targeting using this approach. Antibody was biotinylated to various degrees (1-25 biotins per antibody). Protein stoichiometry of the 2 steps was studied over a range of 2 logs. Both steps, i.e., the biotinylated-antibody (1st step) and streptavidin (2nd step) were radiolabelled (125I and 131I, respectively). A 24-hr interval between 1st and 2nd step was studied, animals being killed 24 hr after the 2nd step. Streptavidin excess led to a decrease in levels of monobiotinylated-antibody in the circulation and in the tumour. Biotinylated-antibody excess led to an increase in circulating levels of streptavidin, a decrease in renal uptake of streptavidin and increased targeting of streptavidin to tumour. At a constant protein molar ratio of biotinylated antibody to streptavidin of 10:1, increasing biotinylation density resulted in an increase in circulating levels, increase in tumour uptake, decrease in renal uptake and increase in liver uptake of streptavidin. As early as 24 hr, the tumour-to-blood ratios of streptavidin already exceeded 1 (max 1.27). Compared with antibody tumour-to-blood ratios, they were better by a factor of between 2 and 3. Tumour-to-normal tissue ratios of radiolabelled streptavidin (with the exception of liver) were also significantly improved when polybiotinylated-antibody was administered first. We have thus shown that the 2-step biotinylated antibody/streptavidin approach can lead to a significant increase in absolute amounts of activity in the tumour under appropriate stoichiometric conditions. This was accompanied by high levels of circulating streptavidin and relatively favourable tumour-to-blood and normal tissue ratios of streptavidin.

Animals↗
PubMed1999

Assay for IkappaB kinases using an in vivo biotinylated IkappaB protein substrate.

IkappaB kinases (IKK)-1 and -2 are related kinases that are induced by stimuli such as TNF or IL-1 to phosphorylate serines 32 and 36 of IkappaBalpha, the regulatory subunit of the transcription factor NF-kappaB. A procedure for an IKK protein kinase assay is described that uses an in vivo biotinylated IkappaB protein substrate, [gamma-(33)P]ATP, and capture onto a streptavidin membrane. Residues 1-54 of the IkappaBalpha substrate were expressed as a fusion with glutathione S-transferase (GST) and a short (22 amino acid) biotinylation sequence that allowed modification during bacterial expression. Using the streptavidin capture assay the phosphorylation activities of recombinant IKK-1 and -2 were characterized. The assay provided a convenient way to compare IKK protein and peptide substrate preferences; biotinylated GST-IkappaBalpha(1-54) was more readily phosphorylated by both IKK-1 and IKK-2 compared to biotinylated myelin basic protein or a 20-mer biotinylated peptide containing serines 32 and 36 of IkappaBalpha. IKK-1 had 83-fold less activity than IKK-2, and the IKK-1+2 complex had approximately 2-fold more activity than IKK-2. IKK-1+2 and IKK-2 had similar K(m) values for ATP and GST-biotin-IkappaB(1-54) and were similarly inhibited by staurosporine and two of its analogues K252a and K252b, suggesting that most of the IkappaBalpha kinase activity in the IKK-1+2 complex may be attributed to IKK-2. Several features of the assay including the broad linear binding range of the streptavidin membranes for the protein substrate GST-biotin-IkappaB(1-54) (1-4000 pmol of protein/cm(2)), the low background, and its capacity for both biotinylated peptides and proteins make it a useful tool for quantitating IKK activity. These factors and the ease of expressing in vivo biotinylated GST fusions will make this assay approach suitable for a wide variety of protein kinases.

Biotin↗
PubMed2001

Metabolic biotinylation of secreted and cell surface proteins from mammalian cells.

Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of medical and scientific applications ranging from drug targeting to immunohistochemistry. To maximize the application of this technology in mammalian systems, we recently demonstrated the ability to metabolically biotinylate tagged proteins in mammalian cells using the endogenous biotin ligase enzymes of the mammalian cell. This technology allows site-specific biotinylation without any exogenous reagents and eliminates possible inactivation of the protein of interest by nonspecific biotinylation. Here, we report further expansion of the mammalian metabolic biotinylation technology to enable biotinylation of proteins secreted from mammalian cells and expressed on their cell surface by cosecretion with BirA, the biotin ligase of E. coli. This technique can be used to biotinylate secreted proteins for purification or targeting and also for biotinylating the surfaces of mammalian cells to facilitate their labeling and purification from other nontagged cells.

Animals↗
PubMed2005

Biotinylation of heat shock protein 70 induces RANTES production in HEK293 cells in a CD40-independent pathway.

Biotinylated proteins and peptides have been used as popular ligands for characterization of cell surface receptors by a variety of methods including flow cytometry. The number and the location of biotin moieties incorporated could alter the structural and physicochemical properties of ligands, although biotin is thought to be such a small molecule (244Da) that it is capable of being conjugated to most proteins without affecting their activity. Here, we demonstrate that the biotinylated HSP70 molecule via primary amines bound to epithelium-like HEK 293 cells in a saturable manner whereas the unlabeled counterparts of HSP70 other than mouse Hsp72 do not. This binding was not competed by either HSP70 or the biotin entity itself. Interestingly, the biotinylated HSP70 also elicited the production of CC-chemokine RANTES independent of CD40 signaling. This response occurred regardless of sequence diversity of HSP70 derived from different species, and neither the biotinylated ovalbumin nor the unlabeled HSP70 cross-linked with a biotinylated protein stimulated a significant level of RANTES production which was induced by biotinylated HSP70 itself. Our findings suggest that modification of HSP70 such as biotinylation may function as a biological alarm signal in the innate immune system.

Biotin↗
PubMed2006

An in vitro method for selective detection of free monomeric ubiquitin by using a C-terminally biotinylated form of ubiquitin.

In an effort to design a selective assay allowing detection of free monomeric ubiquitin, an approach based on a C-terminally biotinylated form of ubiquitin is proposed. In the form of a polyubiquitin chain, ubiquitin marks proteins for degradation by the 26S proteasome. This covalently attached signal is assembled from multiple ubiquitins linked to each other via the C-terminus of one ubiquitin and the epsilon-amine of Lys48 of another ubiquitin. In the present study, a form of ubiquitin having the C-terminus modified with the addition of a biotinylation peptide tag was prepared. After expression, this modified ubiquitin was biotinylated in vitro using recombinant biotin ligase. Biotinylated ubiquitin was further purified using affinity chromatography on immobilized monovalent avidin. This tagged form of ubiquitin is blocked at the C-terminus and therefore can only act as an acceptor (Lys-48 donor) in polyubiquitin chain synthesis. In vitro enzymatic assembly of multiubiquitin chains from biotinylated monoubiquitin and natural monoubiquitin is demonstrated by Western blot analysis using horseradish peroxidase-conjugated streptavidin. Data obtained with this assay indicate potential uses of the C-terminally biotinylated form of ubiquitin for selective detection of monoubiquitin contamination in a cell extract experimentally depleted of ubiquitin, i.e. lysate Fraction II. Cell-free systems established for in vitro examination of ubiquitin involvement in proteolytic processes usually employ Fraction II, which should be essentially ubiquitin-free. It is suggested that the assay using biotinylated monoubiquitin can be useful to exclude the possibility that ubiquitin contamination of laboratory prepared lysate Fraction II accounts for protein degradation in this fraction.

Animals↗
PubMed2004

Production of a biotinylated single-chain antibody fragment in the cytoplasm of Escherichia coli.

Biotinylated antibodies are commonly used reagents in research and molecular diagnostics. The traditional approach to biotinylate antibodies is to conjugate a chemically active biotin derivative to certain chemical groups on protein surface. An alternative method, which can be used for site-specific biotinylation of recombinant antibodies, takes advantage of the capability of the enzyme biotin ligase to catalyze the attachment of a biotin to a unique lysine residue in specific protein/peptide substrates that can be genetically linked to the antibody to generate a fusion protein. We describe here expression of functional scFv and concomitant enzymatic biotinylation of it in bacterial cytoplasm. The anti-thyroid-stimulating hormone (TSH) scFv was produced as an N-terminal fusion with the biotinylated domain of the biotin carboxyl carrier protein of Escherichia coli in the redox modified E. coli strain Origami B which has an oxidizing cytoplasmic environment. After optimization of the biotin concentration and expression temperature, this approach allowed the production of biotinylated and immunoreactive fusion protein with the yield of 1.4 mg/l/OD(600) (13.6 mg/l) in a simple shake flask culture. The biotinylated fusion protein released from disrupted cells can be directly used, for example, in immunoassay applications. This was proved by setting up a TSH immunoassay using the bio-scFv as a solid-phase capture antibody. The sensitivity of the assay was comparable with the currently used commercial immunoassays.

Biotinylation↗
PubMed2004

Development of immunofiltration assay by light addressable potentiometric sensor with genetically biotinylated recombinant antibody for rapid identification of Venezuelan equine encephalitis virus.

A genetically biotinylated single chain fragment variable antibody (scFv) against Venezuelan equine encephalitis virus (VEE) was applied in a system consisting of an immunofiltration enzyme assay (IFA) with a light addressable potentiometric sensor (LAPS) for the rapid identification of VEE. The IFA involved formation of an immunocomplex sandwich consisting of VEE, biotinylated antibody, fluoresceinated antibody and streptavidin, capture of the sandwich by filtration on biotinylated membrane, and labeling of the sandwich by anti-fluorescein urease conjugate. The concentration ratio of biotinylated to fluoresceinated antibodies was investigated and optimized. By the IFA/LAPS assay, the limit of detection (LOD) of VEE was approximately 30 ng/ml, similar to that achieved when chemically biotinylated monoclonal antibody (mAb) was applied. Total assay variance of the IFA/LAPS assay for both intra- and inter-assay precision was less than 20%. Assay accuracy was measured by comparing VEE concentrations estimated by IFA/LAPS standard curve to those obtained by conventional protein assay. VEE concentrations were found to differ by no more than 10%. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzyme-linked immunosorbent assay (ELISA) utilizing polystyrene plates and a chromogenic substrate; however, less time and effort were required for performance of the IFA/LAPS assay. More importantly, use of genetically biotinylated scFv in the IFA/LAPS assay obviates the need for chemical biotinylation of antibody with resultant possible impairment of the antigen-binding site. Furthermore, the potential for batch-to-batch variability resulting from inequality in the number of biotin molecules labeled per antibody molecule is eliminated.

Antibodies, Monoclonal↗
PubMed2006

Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2.

A biotin-protein ligase recognition site (BRS) was inserted into a polypeptide comprised of the maltose-binding protein, the peripheral cannabinoid receptor (CB2), thioredoxin A, and a polyhistidine tag at the carboxy terminus. Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were approximately 1mg per liter of bacterial culture. The biotinylated CB2-fusion fully retained its ligand-binding capacity. Introduction of the BRS at the C-terminus of the CB2 fusion protein (construct CB2-109) resulted in its complete in vivo biotinylation; the biotinylated protein was streptavidin-binding competent. Positioning of the BRS near the N-terminus of CB2 (CB2-112) resulted in a very low level of biotinylation in vivo. However, the detergent solubilized and purified CB2-112 fusion protein were successfully biotinylated in vitro by action of a BirA biotin-protein ligase. The biotinylated CB2-112 fusion protein was cleaved by the tobacco etch virus protease at specifically inserted sites, and deposited onto monomeric avidin agarose beads. Biotinylation of the recombinant CB2 receptor enabled not only purification but also immobilization of the GPCR on a solid support in homogeneous orientation which is beneficial for subsequent structural characterization.

Binding, Competitive↗
PubMed2004

Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels.

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.

Binding Sites↗
PubMed2003

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.

Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

Animals↗
PubMed2001

The biotinyl domain of Escherichia coli acetyl-CoA carboxylase. Evidence that the "thumb" structure id essential and that the domain functions as a dimer.

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme that catalyzes the first committed step of fatty acid synthesis. Similar proteins are found in other bacteria and in chloroplasts. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin to a specific lysine residue. However, the BCCP biotinyl domain differs from many of these proteins in that an eight-amino acid residue insertion is present upstream of the biotinylated lysine. X-ray crystallographic and multidimensional NMR studies show that these residues constitute a structure that has the appearance of an extended thumb that protrudes from the otherwise highly symmetrical domain structure. I report that expression of two mutant BCCPs lacking the thumb residues fails to restore growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperature. Alignment of BCCPs from various organisms shows that only two of the eight thumb residues are strictly conserved, and amino acid substitution of either residue results in proteins giving only weak growth of the temperature-sensitive E. coli strain. Therefore, the thumb structure is essential for the function of BCCP in the ACC reaction and provides a useful motif for distinguishing the biotinylated proteins of multisubunit ACCs from those of enzymes catalyzing other biotin-dependent reactions. An unexpected result was that expression of a mutant BCCP in which the biotinylated lysine residue was substituted with cysteine was able to partially restore growth and fatty acid synthesis to the temperature-sensitive E. coli strain. This complementation was shown to be specific to BCCPs having native structure (excepting the biotinylated lysine) and is interpreted in terms of dimerization of the BCCP biotinyl domain during the ACC reaction.

Acetyl-CoA Carboxylase↗