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Excluded Volume and Weak Interactions in Crowded Solutions Modulate Conformations and RNA Binding of an Intrinsically
Madison A Stringer1,2, Jasmine Cubuk1,2, J Jeremías Incicco1
1Department of Biochemistry and Molecular Biophysics, Washington University in St Louis, 660 Euclid Avenue, Saint Louis, Missouri 63110, United States.
The Journal of Physical Chemistry. B
|June 22, 2023
Summary
Cellular crowding affects protein structure and RNA binding. High-molecular-weight PEG collapses disordered protein tails, while low-molecular-weight PEG expands them, influencing SARS-CoV-2 nucleocapsid protein interactions.
Area of Science:
- Biophysics
- Structural Biology
- Molecular Biology
Background:
- The cellular environment is crowded with macromolecules, influencing protein behavior.
- Intrinsically disordered proteins are particularly sensitive to cellular crowding.
- The SARS-CoV-2 nucleocapsid protein plays a crucial role in viral RNA packaging.
Purpose of the Study:
- To investigate the impact of cellular crowding on the intrinsically disordered N-terminal tail of the SARS-CoV-2 nucleocapsid protein.
- To analyze how crowding affects the interaction between the nucleocapsid protein and RNA.
- To understand the role of crowding in modulating differences between nucleocapsid protein variants.
Main Methods:
- Mimicking cellular crowding using polyethylene glycol (PEG).
- Employing single-molecule Förster resonance energy transfer (smFRET) to study protein conformations.
- Measuring protein-RNA binding affinities under varying crowding conditions and temperatures.
Main Results:
- High-molecular-weight PEG induced a collapse of the disordered tail, while low-molecular-weight PEG caused expansion.
- Crowding agents increased protein-RNA binding affinity, but the effect was non-monotonic with PEG molecular weight.
- Crowding reduced affinity differences between naturally occurring variants of the SARS-CoV-2 nucleocapsid protein.
Conclusions:
- Cellular crowding significantly impacts intrinsically disordered protein regions through a balance of attractive and repulsive forces.
- Crowding modulates protein-ligand interactions, with implications for viral RNA binding.
- Understanding crowding effects is essential for accurate modeling of protein behavior in vivo and for explaining biological variations.
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