Single-Molecule FRET Reveals the Native-State Dynamics of the IκBα Ankyrin Repeat Domain

JOURNAL OF MOLECULAR BIOLOGY

卷:425

Article

Lamboy, Jorge A.¹  Kim, Hajin^2,3,4  Dembinski, Holly¹  Ha, Taekjip^2,3,4  Komives, Elizabeth A.¹ 

[1] Univ Calif San Diego, Dept Chem & Biochem, La Jolla, CA 92037 USA

[2] Univ Illinois, Ctr Phys Living Cells, Dept Phys, Urbana, IL 61801 USA

[3] Univ Illinois, Inst Genom Biol, Urbana, IL 61801 USA

[4] Univ Illinois, Howard Hughes Med Inst, Urbana, IL 61801 USA

关键词: single-molecule FRET;    ankyrin repeat;    intrinsically disordered protein;   

DOI  :  10.1016/j.jmb.2013.04.015

来源: Elsevier

【 摘 要 】

Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of I kappa B alpha were labeled with FRET pairs showed slow fluctuations as if the I kappa B alpha 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 I kappa B alpha 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 degrees 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 I kappa B alpha, 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. (C) 2013 Elsevier Ltd. All rights reserved.

【 授权许可】

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