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Diffusive protein interactions in human versus bacterial cells
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-9848-6357
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-9616-6552
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2020 (English)In: Current Research in Structural Biology, E-ISSN 2665-928X, Vol. 2, p. 68-78Article in journal (Refereed) Published
Abstract [en]

Random encounters between proteins in crowded cells are by no means passive, but found to be under selective control. This control enables proteome solubility, helps to optimise the diffusive search for interaction partners, and allows for adaptation to environmental extremes. Interestingly, the residues that modulate the encounters act mesoscopically through protein surface hydrophobicity and net charge, meaning that their detailed signatures vary across organisms with different intracellular constraints. To examine such variations, we use in-cell NMR relaxation to compare the diffusive behaviour of bacterial and human proteins in both human and Escherichia coli cytosols. We find that proteins that ‘stick’ in E. coli are generally less restricted in mammalian cells. Furthermore, the rotational diffusion in the mammalian cytosol is less sensitive to surface-charge mutations. This implies that, in terms of protein motions, the mammalian cytosol is more forgiving to surface alterations than E. coli cells. The cellular differences seem not linked to the proteome properties per se, but rather to a 6-fold difference in protein concentrations. Our results outline a scenario in which the tolerant cytosol of mammalian cells, found in long-lived multicellular organisms, provides an enlarged evolutionary playground, where random protein-surface mutations are less deleterious than in short-generational bacteria.

Place, publisher, year, edition, pages
2020. Vol. 2, p. 68-78
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-175631DOI: 10.1016/j.crstbi.2020.04.002ISI: 000658373100007Scopus ID: 2-s2.0-85096580569OAI: oai:DiVA.org:su-175631DiVA, id: diva2:1368398
Available from: 2019-11-07 Created: 2019-11-07 Last updated: 2022-12-09Bibliographically approved
In thesis
1. Protein stability and mobility in live cells: Revelation of the intracellular diffusive interaction organization mechanisms
Open this publication in new window or tab >>Protein stability and mobility in live cells: Revelation of the intracellular diffusive interaction organization mechanisms
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 67
Keywords
diffusive interactions, macromolecular crowding, protein thermodynamic stability, protein mobility, in-cell NMR, Hofmeister series
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-175632 (URN)978-91-7797-931-9 (ISBN)978-91-7797-932-6 (ISBN)
Public defence
2019-12-19, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

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.

Available from: 2019-11-26 Created: 2019-11-07 Last updated: 2022-02-26Bibliographically approved
2. How transient interactions in the crowded cytosol affect protein mobility and stability
Open this publication in new window or tab >>How transient interactions in the crowded cytosol affect protein mobility and stability
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most biochemical reactions have evolved in crowded intracellular environments. However, the complexity of intracellular environments is often neglected in structural or functional studies of proteins. In these cases, reactions involving proteins are deliberately separated from the perturbations of co-solutes in order to simplify data acquisition and interpretation. Having acquired an enormous body of knowledge under these simplified dilute-buffer conditions, methodological progress of the past two decades has made the study of proteins inside living cells increasingly accessible and concomitantly kindled an interest to investigate proteins in their native habitat. Naturally, major questions that arose were to what extent ubiquitous transient interactions alter protein structure, function and thermodynamics and, not least, what role protein surfaces and their physicochemical properties play in determining the frequency and duration of these diffusive encounters.

By looking at the rotational-tumbling rates of three structurally well-characterized proteins in live cells with nuclear magnetic resonance (NMR) relaxation, we expand on previous research performed in the bacterium Escherichia coli and establish the physicochemical principles that determine diffusive interactions in the mammalian cytosol of the human ovarian cancer cell line A2780. Just as in E. coli, net charge is the dominating factor in regulating protein interactivity, albeit with the impact on rotational retardation greatly diminished. We ascribe this to the generally lower macromolecular concentrations in the eukaryotic cytosol, and put forward a hypothesis in which less stringent rules regarding protein surface decoration in eukaryotes could have facilitated the development of multi-cellular organisms. Furthermore, by developing a model where a distribution of differently sized interaction partners is taken into account when examining rotational retardation, we reconcile transverse and longitudinal in-cell relaxation with theory, and are able to estimate the populations of the bound and free form of a set of reporter proteins. Looking at the populations of bound protein instead of a mean-field rotational retardation finally allows us to re-assess the guiding rules behind diffusive cytosolic interactions. Last, we outline a putative mechanism behind the in-cell destabilization of a variant of Superoxide dismutase 1 (SOD1barrel). By mimicking generic poly-anionic intracellular co-solutes with poly-acetic acid (NaPAc1200), we identify the positively charged N-terminal portion of the unfolded form of the protein as the interaction site with the highest affinity. Further examining the unfolded ensemble of SOD1barrel with a mutationally destabilized variant reveals a compact state, that remains almost unchanged upon binding to NaPAc1200. This suggests that NaPAc1200-mediated destabilization occurs mainly through mass action, in full accord with the postulated mechanism for in-cell protein destabilization.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 42
Keywords
diffusive transient interactions, macromolecular crowding, protein stability, in-cell NMR, protein evolution
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-185866 (URN)978-91-7911-302-5 (ISBN)978-91-7911-303-2 (ISBN)
Public defence
2020-11-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2020-11-04 Created: 2020-10-14 Last updated: 2022-02-25Bibliographically approved
3. Biophysical chemistry of the ALS-associated protein SOD1: Implications for folding, aggregation and in-cell behaviour
Open this publication in new window or tab >>Biophysical chemistry of the ALS-associated protein SOD1: Implications for folding, aggregation and in-cell behaviour
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biophysical chemistry deals with the structural behavior, properties and molecular function of biological macromolecules. A long-standing challenge is here to establish how these macromolecular features change upon transfer from simplified conditions in vitro to the crowded and molecularly complex environment of live cells.  

This thesis focuses on establishing a general overview of the structural behavior and interaction properties of the ALS-associated protein superoxide dismutase 1 (SOD1) in its natural cellular environment. Importantly, SOD1 constitutes also a multifaceted model system for the yet poorly understood mechanism of protein-aggregation disease, since it is readily amenable to protein-engineering analysis. The focus is on (i) SOD1 folding, (ii) the modulation of the SOD1 properties induced by intracellular interactions and (iii) the process of SOD1 fibrillation, all of which central to the understanding of the ALS disease mechanism. First, we investigate the biophysical role of the disordered catalytic loops in the apoSOD1 monomer, what is identified as the primary aggregation precursor. The results show that these loops play a pivotal role in modulation the apoSOD1 stability due to the generic Flory-entropy penalty, shedding new light to why this species is biased to be aggregation prone. Second, we target the diffusive interactions between SOD1 and the crowded intracellular environment by in-cell NMR. Our findings are that both the rotational tumbling and in-cell stability are controlled by basic physicochemical rules relating to the SOD1 surface properties. Finally, we analyze the kinetics of the SOD1-aggregation behavior in vitro. The observations confirm that the disordered SOD1 loops indeed accelerate the aggregation process because of their penalty to the apo state stability and show, additionally, that they influence the fibril stability.

The physicochemical cues exposed by this thesis work provide not only fundamental clues to our understanding of protein properties, but shed also new light on disease-promoting properties ALS-associated protein SOD1.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2021. p. 67
Keywords
SOD1 ALS folding in-cell NMR aggregation
National Category
Biochemistry and Molecular Biology Biophysics Physical Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-187932 (URN)978-91-7911-394-0 (ISBN)978-91-7911-395-7 (ISBN)
Public defence
2021-02-12, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B or online via Zoom, public link is available at the department website, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2021-01-20 Created: 2020-12-16 Last updated: 2022-02-25Bibliographically approved
4. Diffusive interactions play an important role in protein stability and mobility: Investigations of the intracellular milieu using in-cell NMR
Open this publication in new window or tab >>Diffusive interactions play an important role in protein stability and mobility: Investigations of the intracellular milieu using in-cell NMR
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are crucial for all cellular life. Every signal received by a cell, and every response to it, is mediated by proteins. Inside cells, these proteins diffusively sample each other’s surfaces, as they travel through the cytoplasm in search of their specific interaction partners. In order to carry out their function, proteins need to navigate this net negatively charged and highly crowded milieu without getting stuck with undesirable partners. How do they achieve this?

Previously published data has shown that the bacterial cytoplasm is governed by physicochemical restrictions: There is a net charge interval within which proteins remain soluble. Meaning, if a protein is too positively charged, it will get stuck to the surrounding molecules. If it is too negatively charged, the intracellular mobility approaches that in water, potentially reducing the chance of the protein finding its functional partner. Using in-cell NMR, we have shown that similar charge-based rules govern the molecular mobility inside human cells. The less crowded human cytoplasm does, however, seem more forgiving than the bacterial counterpart, as proteins that experience restricted mobility inside bacteria seem to move freely inside human cells.

The human and bacterial cytoplasm both have a destabilising effect on the ALS-associated ROS scavenger Superoxide Dismutase 1 (SOD1). Our results show that: Stabilised by electrostatic interactions between the positively charged N-terminal and the negatively charged contents of the cytoplasm, the folding equilibrium is shifted towards the unfolded state. Additionally, in the absence of metals, native metal-coordinating surface-exposed histidine residues also contribute to the intracellular destabilisation of SOD1.

Finally, the unfolded state of SOD1 has been characterised in the absence of chemical denaturants. We show that the unfolded state is more compact than previously anticipated. We hypothesise that the increased compactness is caused by the pre-formation of long-range native-like contacts. This implies that: Not only does the primary structure contain the information required for folding, it also contains information on how the unfolded state needs to organise itself to increase the probability of successful folding.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2022. p. 67
Keywords
in-cell NMR, protein-protein interactions, mobility, stability, thermodynamics, Nuclear Magnetic Resonance, stopped-flow spectroscopy, proteins, SOD1, HAH1, TTHA1718, cells, cytoplasm, humans, bacteria, ions, polyions, electrostatics
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-203952 (URN)978-91-7911-890-7 (ISBN)978-91-7911-891-4 (ISBN)
Public defence
2022-09-09, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B and online via Zoom, public link is available at the department website, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2022-08-17 Created: 2022-04-20 Last updated: 2022-08-04Bibliographically approved

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Leeb, SarahSörensen, ThereseYang, FanXin, MuOliveberg, MikaelDanielsson, Jens

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