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The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (Tm = 107.4 ± 0.3 °C) than avidin (Tm = 83.5 ± 0.1 °C) or bacterial streptavidin (Tm = 75.5 °C). AVR4/5 also exhibits a high affinity for biotin (Kd ≈ 3.6 × 10-14 M) comparable to that of avidin and streptavidin (Kd ≈ 10-15 M). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4]5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed. [ABSTRACT FROM AUTHOR]

Integrin α1β1 is one of four collagen-binding integrins in humans. Collagens bind to the αI domain and in the case of α2I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In α2I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligandbound "open" conformation of α2I differs significantly from the "closed" conformation seen in the structure of apo-α2I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to α1I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of α1I with Mg2+ and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-α1I ("closed" conformation) and a model structure of the α1I ("open" conformation) based on the closely related structure of α2I reveals that the binding site is partially blocked to ligands by Glu255 and Tyr285 in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of α2I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of α1I and shown experimentally for α1I using a fluorescent hydrophobic probe. [ABSTRACT FROM AUTHOR]

Demonstrates that tetrameric chicken avidin can be stabilized by introducing intermonomeric disulfide bridges between its subunits. Covalent bonds' lack of major effects on the biotin binding properties of the respective mutants; Tetrameric integrity maintained by mutant Avd-ccci even in denaturing conditions; Supplementary mutants.