Design and construction of highly stable, protease-resistant chimeric avidins
| Autor: | Thomas K.M. Nyholm, Patrick S. Stayton, Olli H. Laitinen, Vesa P. Hytönen, Juha A. E. Määttä, David E. Hyre, Tuomas Kulomaa, Henri R. Nordlund, Markku S. Kulomaa, Eevaleena J. Porkka, Yael Eisenberg-Domovich, Einari A. Niskanen, Oded Livnah, Tiina Paldanius, Jarno Hörhä |
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| Rok vydání: | 2005 |
| Předmět: |
Models
Molecular Biotin binding Insecta Protein family Protein subunit Recombinant Fusion Proteins Molecular Sequence Data Biotin Biosensing Techniques Biology Protein Engineering Biochemistry Protein Structure Secondary Protein structure Animals Amino Acid Sequence Molecular Biology Thermostability Calorimetry Differential Scanning Sequence Homology Amino Acid Temperature Cell Biology Protein engineering Avidin Recombinant Proteins Protein Structure Tertiary Kinetics Microscopy Fluorescence Mutagenesis Biotinylation Mutation biology.protein Chromatography Gel Thermodynamics Electrophoresis Polyacrylamide Gel Endopeptidase K Baculoviridae Chickens Chromatography Liquid Peptide Hydrolases Protein Binding |
| Zdroj: | The Journal of biological chemistry. 280(11) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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