Rational modification of ligand-binding preference of avidin by circular permutation and mutagenesis.
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.
Määttä
Juha A E
JA
Institute of Medical Technology, University of Tampere, 33014 University of Tampere, Finland.
Airenne
Tomi T
TT
Nordlund
Henri R
HR
Jänis
Janne
J
Paldanius
Tiina A
TA
Vainiotalo
Pirjo
P
Johnson
Mark S
MS
Kulomaa
Markku S
MS
Hytönen
Vesa P
VP
D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001360:Avidin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001391:Azo Compounds / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D020134:Catalytic Domain; D002645:Chickens; D018360:Crystallography, X-Ray; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics*; D011489:Protein Denaturation; D017384:Sequence Deletion / Q000235:genetics*; D013379:Substrate Specificity; D013816:Thermodynamics; D044366:Transition Temperature
D030161:Avian Proteins; D001391:Azo Compounds; D008024:Ligands; D001360:Avidin; C001413:HABA; D001710:Biotin