Rational modification of ligand-binding preference of avidin by circular permutation and mutagenesis

Juha A. E. Määttä, Tomi Airenne, Henri R. Nordlund, Janne Jänis, T Paldanius, Pirjo Vainiotalo, Mark Johnson, Markku S. Kulomaa, Vesa P. Hytönen

Tutkimustuotos: LehtiartikkeliArtikkeliTieteellinenvertaisarvioitu

Abstrakti

Chicken avidin is a key component used in a wide variety of biotechnological applications. Here we present a circularly permuted avidin (cpAvd4-->3) that lacks the loop between beta-strands 3 and 4. Importantly, the deletion of the loop has a positive effect on the binding of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) to avidin. To increase the HABA affinity of cpAvd4-->3 even further, we mutated asparagine 118 on the bottom of the ligand-binding pocket to methionine, which simultaneously caused a significant drop in biotin-binding affinity. The X-ray structure of cpAvd4--> 3(N118M) allows an understanding of the effect of mutation to biotin-binding, whereas isothermal titration calorimetry revealed that the relative binding affinity of biotin and HABA had changed by over one billion-fold between wild-type avidin and cpAvd4-->3(N118M). To demonstrate the versatility of the cpAvd4-->3 construct, we have shown that it is possible to link cpAvd4-->3 and cpAvd5-->4 to form the dual-chain avidin called dcAvd2. These novel avidins might serve as a basis for the further development of self-organising nanoscale avidin building blocks.
AlkuperäiskieliEnglanti
Sivut1124-35
JulkaisuChemBioChem
Vuosikerta9
Numero7
TilaJulkaistu - 5 toukok. 2008
OKM-julkaisutyyppiA1 Julkaistu artikkeli, soviteltu

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