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Thermodynamics of protein destabilization in live cells
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.ORCID-id: 0000-0002-6048-6896
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Vise andre og tillknytning
Rekke forfattare: 112015 (engelsk)Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, nr 40, s. 12402-12407Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a beta-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 degrees C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized interaction landscape of the cellular interior.

sted, utgiver, år, opplag, sider
2015. Vol. 112, nr 40, s. 12402-12407
Emneord [en]
thermodynamics, protein stability, crowding, in vivo, NMR
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-123537DOI: 10.1073/pnas.1511308112ISI: 000363125400053OAI: oai:DiVA.org:su-123537DiVA, id: diva2:874590
Tilgjengelig fra: 2015-11-27 Laget: 2015-11-27 Sist oppdatert: 2019-12-12bibliografisk kontrollert
Inngår i avhandling
1. Protein stability and mobility in live cells: Revelation of the intracellular diffusive interaction organization mechanisms
Åpne denne publikasjonen i ny fane eller vindu >>Protein stability and mobility in live cells: Revelation of the intracellular diffusive interaction organization mechanisms
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Biochemical processes inside living cells take place in a confined and highly crowded environment. As such, macromolecular crowding, one of the most important physicochemical properties of cytoplasm, is an essential element of cell physiology. It not only gives rise to steric repulsion, but also promotes non-specific, transient, interactions (referred to as diffusive interactions) between molecules. Since diffusive interactions are a key way to achieving a highly organized intracellular environment, without such interactions, the cell is just “a bag of molecules”. Therefore, understanding how diffusive interactions modulate protein behavior in live cells is of fundamental importance for revealing the mechanisms of molecular recognition, as well as for understanding the cause of protein misfolding diseases.

This thesis focuses on how macromolecular crowding influences the stability and diffusive motions of proteins within living cells by modulating their diffusive interactions. First, we investigated the thermal stability of superoxide dismutase 1 (SOD1), a protein involved in the development of familial amyotrophic lateral sclerosis (ALS), in mammalian and E. coli cells. Intriguingly, the major thermodynamic consequence of macromolecular crowding is due not only to conventional steric repulsions, but primarily to the detailed chemical nature of the diffusive protein interactions in live cells. Secondly, we presented a mutational study of how these diffusive interactions influence the rotation of proteins in the mammalian and bacterial cytosol. The result is a quantitative description of the physicochemical code for the intracellular protein motion, showing that it depends critically on the surface details of protein and the type of the host cell as well. Thirdly, we characterized the impact of  intracellular protein concentration by altering the volume of E. coli cells by osmotic shock. The results obtained show that the intracellular diffusion of proteins is not determined by the chemical properties of the protein surface alone, but also by the frequency of concentration-dependent encounters. Moreover, it appears that eukaryotes and bacteria have achieved fidelity of biological processes through different evolutionary strategies. Overall, these observations have numerous implications for both functional protein design and deciphering the evolution of the surface characteristics of proteins.

Subsequently, we attempted to shed new light on the Hofmeister series, using protein-folding kinetics as observable. The results indicate that the Hofmeister series cannot be explained entirely by the traditional Kosmotropes/Chaotropes classification. Strong hetero-ion pairing cannot be ignored when trying to understand the effects of salts on protein salting-in and salting-out behaviors.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. s. 67
Emneord
diffusive interactions, macromolecular crowding, protein thermodynamic stability, protein mobility, in-cell NMR, Hofmeister series
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-175632 (URN)978-91-7797-931-9 (ISBN)978-91-7797-932-6 (ISBN)
Disputas
2019-12-19, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.

Tilgjengelig fra: 2019-11-26 Laget: 2019-11-07 Sist oppdatert: 2019-11-19bibliografisk kontrollert

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