Thermodynamics of the binding of biotin and some analogues by avidin.
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ABSTRACT:
SUBMITTER: Green NM
PROVIDER: S-EPMC1270186 | biostudies-other | 1966 Dec
REPOSITORIES: biostudies-other
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Project description:The binding of biotin to avidin is one of the strongest in nature with absolute free energy of binding, ?A(0) = -20.4 kcal/mol. Therefore, this complex became a target for a large number of computational studies, which all, however, are based on approximate techniques or simplified models and have led to a wide range of results Therefore, ?A(0) is calculated here by rigorous statistical mechanical methods and models that consider long-range electrostatics. (1) We apply our method, "hypothetical scanning molecular dynamics with thermodynamic integration" (HSMD-TI) to avidin-biotin modeled by periodic boundary conditions with particle mesh ewald (PME). (2) We apply the double decoupling method (DDM) to this system modeled by the spherical solvent boundary potential (SSBP) and the generalized solvent boundary potential (GSBP). The corresponding results for neutral biotin, ?A(0) = -29.1 ± 0.8 and -25.2 ± 0.5 kcal/mol are significantly lower than the experimental value; we also provide the result for a charged biotin, ?A(0) = -33.3 ± 0.8 kcal/mol. It is plausible to suggest that this disagreement with the experiment may stem from ignoring the (positive) contribution of a mobile loop that changes its structure upon ligand binding.
| S-EPMC3383089 | biostudies-literature
Project description:The high affinity (KD ~ 10-15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4'-hydroxyazobenzene)-benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values revealed strong temperature dependence with large activation energies (6-15 kcal/mol) that do not correspond to a diffusion limited process (3-4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications.
| S-EPMC6394990 | biostudies-other
Project description:BackgroundA chicken egg contains several biotin-binding proteins (BBPs), whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings.ResultsTwo separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin.ConclusionWe describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.
| S-EPMC1082904 | biostudies-literature
Project description:The object of this study was to define minimized biotin-binding fragments, or 'prorecognition sites', of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin.
| S-EPMC1151383 | biostudies-other
Project description:Affinity interactions between the small molecule biotin and the protein avidin have been used extensively to functionalize biomaterials. More recently, researchers have leveraged the changes in biotin-avidin affinity that occur upon biotin conjugation to larger molecules to control the release of biotinylated drugs and proteins. However, the effects of biotin-avidin interactions on hydrogel properties have not been thoroughly investigated. The objective of this study was to evaluate the effect of increasing biotin and avidin concentrations on hydrogel swelling properties, as an indicator of crosslinking. Gelatin, selected as a model hydrogel material, was biotinylated at increasing fold molar excesses of biotin with a PEG linker using N-hydroxysuccinimide chemistry. Afterwards, biotinylated gelatin was formed into hydrogels and stabilized with glutaraldehyde. Swelling properties of the biotinylated hydrogels were investigated by conducting swelling studies in different avidin solutions. Increasing the degree of biotinylation caused significant decreases in swelling ratios of the hydrogels in a dose-dependent manner, suggesting increases in crosslinking of the hydrogels. However, increasing avidin concentrations in excess of biotin content did not significantly affect swelling ratios. Moving hydrogels to phosphate-buffered saline following avidin incorporation resulted in increased swelling ratios for hydrogels prepared with a lower concentration of biotin. However, hydrogels prepared with the highest concentration of biotin did not experience increased swelling ratios, implying that the stability of biotin-avidin-mediated crosslinking depends on the number of biotin molecules available for binding. Collectively, these results demonstrate that biotin-avidin interactions control hydrogel swelling properties, and that the magnitude and stability of the effects depend on the biotin concentration. These results have important implications for affinity-based controlled release of biotinylated drugs or proteins from biotin-avidin-crosslinked hydrogels.
| S-EPMC7711042 | biostudies-literature
Project description:In this work the synthesis, photochemistry, and streptavidin interaction of new [Ru(tpy)(bpy)(SRR')](PF6)2 complexes where the R' group contains a free biotin ligand, are described. Two different ligands SRR' were investigated: An asymmetric ligand 1 where the Ru-bound thioether is a N-acetylmethionine moiety linked to the free biotin fragment via a triethylene glycol spacer and a symmetrical ligand 2 containing two identical biotin moieties. The coordination of these two ligands to the precursor [Ru(tpy)(bpy)Cl]Cl was studied in water at 80 °C. In such conditions the coordination of the asymmetric ligand 1 occurred under thermodynamic control. After the reaction, a mononuclear and a binuclear complex were isolated. In the mononuclear complex, the ratio of methionine- {[6](PF6)2} vs. biotin-bound {[7](PF6)2} regioisomer was 5.3 and the free biotin fragment of [6](PF6)2 allowed to purify it from its isomer [7](PF6)2 at small scales using avidin affinity chromatography. Coordination of the symmetrical ligand 2 afforded [Ru(tpy)(bpy)(2)](PF6)2 {[8](PF6)2} in synthetically useful scales (100 mg), good yield (82 %), and without traces of the binuclear impurity. In this complex, one of the biotin remains free whereas the second one is coordinated to ruthenium. Photochemical release of ligand 2 from [8](PF6)2 occurred upon blue light irradiation (465 nm) with a photosubstitution quantum yield of 0.011 that was independent of the binding of streptavidin to the free biotin ligand.
| S-EPMC6473509 | biostudies-literature
Project description:Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin-biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys(111)-->isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin-biotin technological applications.
| S-EPMC1222514 | biostudies-other
Project description:The tight interaction between the vitamin biotin and the protein avidin is so strong (Ka approximately 10(15) M-1) that conditions which are usually sufficient for protein denaturation fail to dissociate the avidin-biotin complex. In order to form a reversible interaction between the two biomolecules, we have modified the binding-site tyrosine by nitration, thus reducing the pKa of the phenol group which forms a crucial hydrogen bond with the ureido group of biotin. At relatively low pH values (4-5), the resultant modified forms of avidin bind biotin with a very high association constant ( > 10(9) M-1). The modified avidins are thus capable of supporting stable, long-term binding of biotin or biotinylated macro-molecules. The latter molecules can be detached by increasing the pH of the medium or by introduction of excess levels of biotin at neutral pH. These findings demonstrate the importance of a single hydrogen bond for strong biotin binding. The new derivatives of avidin should be useful for applications whereby a reversible interaction between the four biotin-binding sites and biotin is desired, thus increasing the versatility of the avidin-biotin system for biotechnological application.
| S-EPMC1217322 | biostudies-other
Project description:Developing magnetic resonance imaging (MRI) contrast agents with high relaxivity and specificity was essential to increase MRI diagnostic sensitivity and accuracy. In this study, the MRI contrast agent, vascular endothelial growth factor receptor (VEGFR)-targeted poly (l-lysine) (PLL)-diethylene triamine pentacetate acid (DTPA)-gadolinium (Gd) (VEGFR-targeted PLL-DTPA-Gd, VPDG), was designed and prepared to enhance the MRI diagnosis capacity of tumor. Biotin-PLL-DTPA-Gd was synthesized first, then, VEGFR antibody was linked to biotin-PLL-DTPA-Gd using biotin-avidin reaction. In vitro cytotoxicity study results showed that VPDG had low toxicity to MCF-7 cells and HepG2 cells at experimental concentrations. In cell uptake experiments, VPDG could significantly increase the internalization rates (61.75%±5.22%) in VEGFR-positive HepG2 cells compared to PLL-DTPA-Gd (PDG) (25.16%±4.71%, P<0.05). In MRI studies in vitro, significantly higher T1 relaxivity (14.184 mM-1 s-1) was observed compared to Magnevist® (4.9 mM-1 s-1; P<0.01). Furthermore, in vivo MRI study results showed that VPDG could significantly enhance the tumor signal intensity and prolong the diagnostic time (from <1 h to 2.5 h). These results indicated that macromolecular VPDG was a promising MRI contrast agent and held great potential for molecular diagnosis of tumor.
| S-EPMC5523973 | biostudies-literature
Project description:The unusually strong reversible binding of biotin by avidin and streptavidin has been investigated by density functional and MP2 ab initio quantum mechanical methods. The solvation of biotin by water has also been studied through QM/MM/MC calculations. The ureido moiety of biotin in the bound state hydrogen bonds to five residues, three to the carbonyl oxygen and one for each--NH group. These five hydrogen bonds act cooperatively, leading to stabilization that is larger than the sum of individual hydrogen-bonding energies. The charged aspartate is the key residue that provides the driving force for cooperativity in the hydrogen-bonding network for both avidin and streptavidin by greatly polarizing the urea of biotin. If the residue is removed, the network is disrupted, and the attenuation of the energetic contributions from the neighboring residues results in significant reduction of cooperative interactions. Aspartate is directly hydrogen-bonded with biotin in streptavidin and is one residue removed in avidin. The hydrogen-bonding groups in streptavidin are computed to give larger cooperative hydrogen-bonding effects than avidin. However, the net gain in electrostatic binding energy is predicted to favor the avidin-bicyclic urea complex due to the relatively large penalty for desolvation of the streptavidin binding site (specifically expulsion of bound water molecules). QM/MM/MC calculations involving biotin and the ureido moiety in aqueous solution, featuring PDDG/PM3, show that water interactions with the bicyclic urea are much weaker than (strept)avidin interactions due to relatively low polarization of the urea group in water.
| S-EPMC2527462 | biostudies-literature