Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces.
In this study we showed that tetrameric chicken avidin can be stabilized by introducing intermonomeric disulfide bridges between its subunits. These covalent bonds had no major effects on the biotin binding properties of the respective mutants. Moreover, one of the mutants (Avd-ccci) maintained its tetrameric integrity even in denaturing conditions. The new avidin forms Avd-ci and Avd-ccci, which have native --> denatured transition midpoints (T(m)) of 98.6 and 94.7 degrees C, respectively, in the absence of biotin, will find use in applications where extreme stability or minimal leakage of subunits is required. Furthermore, we showed that the intramonomeric disulfide bridges found in the wild-type avidin affect its stability. The mutant Avd-nc, in which this bridge was removed, had a lower T(m) in the absence of biotin than the wild-type avidin but showed comparable stability in the presence of biotin.
Nordlund
Henri R
HR
Department of Biological and Environmental Science, P. O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Finland.
Laitinen
Olli H
OH
Uotila
Sanna T H
ST
Nyholm
Thomas
T
Hytönen
Vesa P
VP
Slotte
J Peter
JP
Kulomaa
Markku S
MS
D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000235:genetics*; D001710:Biotin / Q000737:chemistry*; D002151:Calorimetry; D002460:Cell Line; D002645:Chickens; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D007313:Insecta; D007532:Isoleucine / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D004220:Disulfides; D007532:Isoleucine; D001360:Avidin; D001710:Biotin; D003545:Cysteine