Chicken Avidin-related Protein 4/5 Shows Superior Thermal Stability when Compared with Avidin while Retaining High Affinity to Biotin

• Published in: The Journal of biological chemistry Vol. 279; no. 10; pp. 9337 - 9343
• Main Authors: Hytönen, Vesa P., Nyholm, Thomas K.M., Pentikäinen, Olli T., Vaarno, Jonne, Porkka, Eevaleena J., Nordlund, Henri R., Johnson, Mark S., Slotte, J. Peter, Laitinen, Olli H., Kulomaa, Markku S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.03.2004; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Avian Proteins - chemistry; Avian Proteins - genetics; Avidin - chemistry; Avidin - genetics; Biotin - chemistry; Chickens; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Denaturation; Protein Engineering; Protein Structure, Quaternary; Recombinant Proteins; Temperature
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M310989200

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 × 10-14m) comparable to that of avidin and streptavidin (Kd ≈ 10-15m). 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.