Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain

Jorge A. Lamboy, Hajin Kim, Holly Dembinski, Taekjip Ha, Elizabeth A. Komives

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)2578-2590
Number of pages13
JournalJournal of molecular biology
Volume425
Issue number14
DOIs
StatePublished - Jul 24 2013
Externally publishedYes

Keywords

  • ankyrin repeat
  • intrinsically disordered protein
  • single-molecule FRET

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain'. Together they form a unique fingerprint.

  • Cite this