title: Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin.
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 degrees C) than avidin (T(m) = 83.5 +/- 0.1 degrees C) or bacterial streptavidin (T(m) = 75.5 degrees C). AVR4/5 also exhibits a high affinity for biotin (K(d) approximately 3.6 x 10(-14) m) comparable to that of avidin and streptavidin (K(d) approximately 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.
authors.affiliation: Department of Biological and Environmental Science, P. O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Finland.
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.forename: Thomas K M
authors.initials: TK
authors.lastname: Nyholm
authors.forename: Olli T
authors.initials: OT
authors.lastname: Pentikäinen
authors.forename: Jonne
authors.initials: J
authors.lastname: Vaarno
authors.forename: Eevaleena J
authors.initials: EJ
authors.lastname: Porkka
authors.forename: Henri R
authors.initials: HR
authors.lastname: Nordlund
authors.forename: Mark S
authors.initials: MS
authors.lastname: Johnson
authors.forename: J Peter
authors.initials: JP
authors.lastname: Slotte
authors.forename: Olli H
authors.initials: OH
authors.lastname: Laitinen
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics; D001360:Avidin / Q000737:chemistry* / Q000235:genetics; D001710:Biotin / Q000737:chemistry; D002645:Chickens; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins; D013696:Temperature
chemical_list: D030161:Avian Proteins; D011994:Recombinant Proteins; D001360:Avidin; D001710:Biotin