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GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study

  • Original Article
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Journal of Chemical Biology

Abstract

The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ∼8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.

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Abbreviations

A ptp :

peak to peak amplitude

EPR:

Electron paramagnetic resonance

FRET:

Fluorescence resonance energy transfer

GuHCl:

Guanidine hydrochloride

HCA II:

Human carbonic anhydrase II

MTSSL:

(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin label

R1:

Cysteine side chain labeled with MTSSL

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Acknowledgements

We thank Professor Anders Lund for giving us access to the EPR lab at IFM-Chemical Physics, Linköping University and Professor Sandra S. Eaton, University of Denver, for advice concerning EPR data analysis. We acknowledge Professor Mikael Lindgren and Dr. Malin Persson for valuable discussions and advice. We also gratefully acknowledge Dr. Christian Altenbach, University of California, Los Angeles, for providing his software for determining inter-residue distances. This work was supported by grants from the Swedish Foundation for Strategic Research (PH), the Swedish Research Council (BHJ, PH, and UC), and the Knut and Alice Wallenberg Foundation (BHJ, PH, and UC). PH is a Royal Swedish Academy of Sciences Research Fellow supported by a grant from the Knut and Alice Wallenberg Foundation.

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Authors and Affiliations

  1. IFM-Department of Chemistry, Linköping University, 58183, Linköping, Sweden

    Rikard Owenius, Uno Carlsson & Per Hammarström

  2. Department of Photochemistry & Molecular Science, Uppsala University, 75120, Uppsala, Sweden

    Rikard Owenius

  3. IFM-Department of Molecular Biotechnology, Linköping University, 58183, Linköping, Sweden

    Anngelica Jarl & Bengt-Harald Jonsson

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  1. Rikard Owenius
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  2. Anngelica Jarl
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  4. Uno Carlsson
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  5. Per Hammarström
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Correspondence to Per Hammarström.

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Owenius, R., Jarl, A., Jonsson, BH. et al. GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study. J Chem Biol 3, 127–139 (2010). https://doi.org/10.1007/s12154-010-0038-2

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