TY - JOUR
T1 - Design and construction of highly stable, protease-resistant chimeric avidins
AU - Hytönen, Vesa P.
AU - Määttä, Juha A.E.
AU - Nyholm, Thomas K.M.
AU - Livnah, Oded
AU - Eisenberg-Domovich, Yael
AU - Hyre, David
AU - Nordlund, Henri R.
AU - Hörhä, Jarno
AU - Niskanen, Einari A.
AU - Paldanius, Tiina
AU - Kulomaa, Tuomas
AU - Porkka, Eevaleena J.
AU - Stayton, Patrick S.
AU - Laitinen, Olli H.
AU - Kulomaa, Markku S.
PY - 2005/3/18
Y1 - 2005/3/18
N2 - 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 (Tm = 106.4°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 (Tm = 96.5°C) than native avidin (Tm = 83.5°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 (Tm = 97.5°C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (Tm = 111.1°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.
AB - 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 (Tm = 106.4°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 (Tm = 96.5°C) than native avidin (Tm = 83.5°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 (Tm = 97.5°C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (Tm = 111.1°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.
UR - http://www.scopus.com/inward/record.url?scp=20144363959&partnerID=8YFLogxK
U2 - 10.1074/jbc.M414196200
DO - 10.1074/jbc.M414196200
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C2 - 15649900
AN - SCOPUS:20144363959
SN - 0021-9258
VL - 280
SP - 10228
EP - 10233
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 11
ER -