title: Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain.
abstract: Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1-4), at ARs 2 and 5 (AR 2-5), and at ARs 3 and 6 (AR 3-6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 "loses grip". AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92037-0378, USA.
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Lamboy
authors.forename: Hajin
authors.initials: H
authors.lastname: Kim
authors.forename: Holly
authors.initials: H
authors.lastname: Dembinski
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Komives
mesh_terms: D019943:Amino Acid Substitution; D017089:Ankyrin Repeat; D031541:Fluorescence Resonance Energy Transfer; D020672:I-kappa B Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D058767:Protein Unfolding
chemical_list: D020672:I-kappa B Proteins; D050505:Mutant Proteins