title: Design and construction of highly stable, protease-resistant chimeric avidins.
abstract: The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.
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: Juha A E
authors.initials: JA
authors.lastname: Määttä
authors.forename: Thomas K M
authors.initials: TK
authors.lastname: Nyholm
authors.forename: Oded
authors.initials: O
authors.lastname: Livnah
authors.forename: Yael
authors.initials: Y
authors.lastname: Eisenberg-Domovich
authors.forename: David
authors.initials: D
authors.lastname: Hyre
authors.forename: Henri R
authors.initials: HR
authors.lastname: Nordlund
authors.forename: Jarno
authors.initials: J
authors.lastname: Hörhä
authors.forename: Einari A
authors.initials: EA
authors.lastname: Niskanen
authors.forename: Tiina
authors.initials: T
authors.lastname: Paldanius
authors.forename: Tuomas
authors.initials: T
authors.lastname: Kulomaa
authors.forename: Eevaleena J
authors.initials: EJ
authors.lastname: Porkka
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
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; D001360:Avidin / Q000138:chemical synthesis* / Q000737:chemistry*; D016367:Baculoviridae / Q000378:metabolism; D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002645:Chickens; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000737:chemistry; D007313:Insecta; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010447:Peptide Hydrolases / Q000494:pharmacology*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013816:Thermodynamics
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001360:Avidin; D001710:Biotin; D010447:Peptide Hydrolases; D019286:Endopeptidase K