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The unfolded ß-barrel of SOD1 is in a compact state, stabilised by long-range hydrophobic contacts.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-6048-6896
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-1919-7520
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The unfolded state of a globular protein in a physiologically relevant environment is by no means an inert random coil.  On the contrary, its structural and dynamic properties are crucial for e.g., protein folding and aggregation.  Despite its importance, it has been studied relatively sparsely, which is partly due to its low population which tend to obstruct detailed biophysical characterization.  Here, introduction of two destabilizing core mutations allow us to study the unfolded state of the central b-barrel of Superoxide Dismutase 1 under native conditions.  

In order to structurally characterise the unfolded state, we use high-resolution nuclear magnetic resonance (NMR), including paramagnetic relaxation enhancement, to obtain constraints for the generation of unfolded ensembles.  The results show that the unfolded state is more compact than the chemically denatured state of the same protein.  This compacted state seems to be stabilised by long-range hydrophobic contacts, out of which many coincide with those found in the native state.  We also investigated the previously observed destabilising effect on the unfolded state by a poly-anion, and find that; the interaction does not alter the overall ensemble dimensions, nor the pattern in native-like contacts.  On the other hand, addition of the chemical denaturant urea results in a more expanded state.  The varying compaction with different co-solutes was validated by pulsed-field gradient NMR diffusion measurements.  

Unlike helical proteins, b-proteins lack the ability to fulfil hydrogen bonds by local native interactions. This forces specific prerequisites on the collapsed pre-folding state.  Here, the compaction is enabled by both native-like and non-native long-range contacts in the unfolded ensemble, and we suggest that the average topology of the collapsed state is determined by the sequence distribution of hydrophobic patches, separated by non-interacting hydrophilic clusters. 

National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-203621OAI: oai:DiVA.org:su-203621DiVA, id: diva2:1652295
Available from: 2022-04-18 Created: 2022-04-18 Last updated: 2022-04-20
In thesis
1. 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, ThereseDanielsson, JensOliveberg, Mikael

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