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High intracellular stability of the spidroin N-terminal domain in spite of abundant amyloidogenic segments revealed by in-cell hydrogen/deuterium exchange mass spectrometry

Kaldmäe, Margit and Leppert, Axel and Chen, Gefei and Sarr, Medoune and Sahin, Cagla and Nordling, Kerstin and Kronqvist, Nina and Gonzalvo‐Ulla, Marta and Fritz, Nicolas and Abelein, Axel and Laín, Sonia and Biverstål, Henrik and Jörnvall, Hans and Lane, David P. and Rising, Anna and Johansson, Jan and Landreh, Michael (2020) High intracellular stability of the spidroin N-terminal domain in spite of abundant amyloidogenic segments revealed by in-cell hydrogen/deuterium exchange mass spectrometry. FEBS Journal, 287 (13). pp. 2823-2833. ISSN 1742-464X.

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Proteins require an optimal balance of conformational flexibility and stability in their native environment to ensure their biological functions. A striking example is spidroins, spider silk proteins, which are stored at extremely high concentrations in soluble form, yet undergo amyloid‐like aggregation during spinning. Here, we elucidate the stability of the highly soluble N‐terminal domain (NT) of major ampullate spidroin 1 in the Escherichia coli cytosol as well as in inclusion bodies containing fibrillar aggregates. Surprisingly, we find that NT, despite being largely composed of amyloidogenic sequences, showed no signs of concentration‐dependent aggregation. Using a novel intracellular hydrogen/deuterium exchange mass spectrometry (HDX‐MS) approach, we reveal that NT adopts a tight fold in the E. coli cytosol and in this manner conceals its aggregation‐prone regions by maintaining a tight fold under crowded conditions. Fusion of NT to the unstructured amyloid‐forming Aβ₄₀ peptide, on the other hand, results in the formation of fibrillar aggregates. However, HDX‐MS indicates that the NT domain is only partially incorporated into these aggregates in vivo . We conclude that NT is able to control its aggregation to remain functional under the extreme conditions in the spider silk gland.

Item Type:Article
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URLURL TypeDescription
Fritz, Nicolas0000-0002-9722-7425
Johansson, Jan0000-0002-8719-4703
Landreh, Michael0000-0002-7958-4074
Alternate Title:Intracellular HDX-MS of a highly soluble spider silk domain
Additional Information:© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Received 26 July 2019, revised 1 November 2019, accepted 5 December 2019. ML is supported by an Ingvar Carlsson Award from the Swedish Foundation for Strategic Research, a KI Faculty‐funded Career Position, a KI‐StratNeuro Starting Grant and a VR Starting Grant (2019‐01961). CS is supported by a Novo Nordisk Foundation Postdoctoral Fellowship (NNF19OC0055700). SL is supported by Barncancerfonden grant TJ2014‐0013. DPL is supported by VR grant 2013_08807. JJ and AR acknowledge support from the Center for Innovative Medicine at Karolinska Institutet (CIMED), VR and Vinnova. ML gratefully acknowledges technical support from MS Vision, NL. The authors declare no conflict of interest. Author contributions: AR, JJ and ML designed the study with input from HJ, SL and DPL. MK, AL, GC and MS expressed and purified protein. MK, MG‐U and ML performed mass spectrometry measurements. CS and NF performed protein characterization. KN, NK, AA and HB provided constructs and reagents. MK, AR, JJ and ML wrote the paper with input from all authors.
Funding AgencyGrant Number
Swedish Foundation for Strategic ResearchUNSPECIFIED
Karolinska InstitutetUNSPECIFIED
NovoNordisk FoundationNNF19OC0055700
Subject Keywords:hydrogen/deuterium exchange mass spectrometry; intracellular protein folding; protein aggregation; spider silk
Issue or Number:13
Record Number:CaltechAUTHORS:20191218-142203346
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Official Citation:Kaldmäe, M., Leppert, A., Chen, G., Sarr, M., Sahin, C., Nordling, K., Kronqvist, N., Gonzalvo‐Ulla, M., Fritz, N., Abelein, A., Laίn, S., Biverstål, H., Jörnvall, H., Lane, D.P., Rising, A., Johansson, J. and Landreh, M. (2020), High intracellular stability of the spidroin N‐terminal domain in spite of abundant amyloidogenic segments revealed by in‐cell hydrogen/deuterium exchange mass spectrometry. FEBS J, 287: 2823-2833. doi:10.1111/febs.15169
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:100361
Deposited By: Tony Diaz
Deposited On:18 Dec 2019 23:06
Last Modified:08 Jul 2020 16:14

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