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J. Biol. Chem., Vol. 279, Issue 10, 9337-9343, March 5, 2004

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

Vesa P. Hytönen, Thomas K. M. Nyholm, Olli T. Pentikäinen, Jonne Vaarno¶, Eevaleena J. Porkka, Henri R. Nordlund, Mark S. Johnson, J. Peter Slotte, Olli H. Laitinen||, and Markku S. Kulomaa**

From the Department of Biological and Environmental Science, P. O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Finland, the Department of Biochemistry and Pharmacy, Åbo Akademi University, P. O. Box 66, FIN-20521 Turku, Finland, the ^¶Arctic Diagnostics Oy, FIN-20521 Turku, Finland, and the ^||A. I. Virtanen Institute, Department of Molecular Medicine, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland

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 °C) than avidin (T_m = 83.5 ± 0.1 °C) or bacterial streptavidin (T_m = 75.5 °C). AVR4/5 also exhibits a high affinity for biotin (K_d 3.6 x 10^-14 M) comparable to that of avidin and streptavidin (K_d 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.

Received for publication, October 6, 2003 , and in revised form, November 11, 2003.

^* This work was supported by the Academy of Finland (to T. K. M. N., O. T. P., J. P. S., and M. S. J.) and the National Graduate School in Informational and Structural Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. E-mail: markku.kulomaa{at}csc.fi.

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