A1 Refereed original research article in a scientific journal
Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin
Authors: 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
Publication year: 2004
Journal: Journal of Biological Chemistry
Journal name in source: JOURNAL OF BIOLOGICAL CHEMISTRY
Journal acronym: J BIOL CHEM
Volume: 279
Issue: 10
First page : 9337
Last page: 9343
Number of pages: 7
ISSN: 0021-9258
DOI: https://doi.org/10.1074/jbc.M310989200(external)
Abstract
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.
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.
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