title: Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces.
abstract: 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.
authors.affiliation: Department of Biological and Environmental Science, P. O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Finland.
authors.forename: Henri R
authors.initials: HR
authors.lastname: Nordlund
authors.forename: Olli H
authors.initials: OH
authors.lastname: Laitinen
authors.forename: Sanna T H
authors.initials: ST
authors.lastname: Uotila
authors.forename: Thomas
authors.initials: T
authors.lastname: Nyholm
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.forename: J Peter
authors.initials: JP
authors.lastname: Slotte
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
mesh_terms: 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
chemical_list: D018076:DNA, Complementary; D004220:Disulfides; D007532:Isoleucine; D001360:Avidin; D001710:Biotin; D003545:Cysteine