Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin

: Hytonen VP, Nyholm TKM, Pentikainen OT, Vaarno J, Porkka EJ, Nordlund HR, Johnson MS, Slotte JP, Laitinen OH, Kulomaa MS

Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

: 2004

: Journal of Biological Chemistry

: JOURNAL OF BIOLOGICAL CHEMISTRY

: J BIOL CHEM

: 279

: 10

: 9337

: 9343

: 7

: 0021-9258

DOI: https://doi.org/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 (T-m = 107.4 +/- 0.3 degreesC) than avidin (T-m = 83.5 +/- 0.1 degreesC) or bacterial streptavidin (T-m = 75.5 degreesC). AVR4/5 also exhibits a high affinity for biotin (K-d approximate to 3.6 x 10(-14) M) comparable to that of avidin and streptavidin (K-d approximate to 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.