Weak, specific chemical interactions dictate barnase stability in diverse cellular environments

Area of Science:

• Biochemistry
• Cell Biology
• Protein Science

Background:

• In vitro studies often fail to replicate in-cell protein behavior due to factors like macromolecular crowding and chemical interactions.
• Small proteins and peptides may be more sensitive to specific chemical interactions than larger proteins within the cell.

Purpose of the Study:

• To investigate how small protein folding is influenced by diverse cellular environments, including different intracellular compartments and host organisms.
• To compare in-cell protein folding dynamics with in vitro models and cellular lysates.

Main Methods:

• Utilized Förster Resonance Energy Transfer (FRET)-labeled barnase as a model small protein.
• Employed fast relaxation imaging to monitor protein folding in live U2-OS cells (cytoplasm and nucleus).
• Compared in-cell observations with in vitro crowding agents (Ficoll, M-PER™) and cellular lysates.

Main Results:

• Barnase folding was stabilized in the cytoplasm and destabilized in the nucleus of U2-OS cells.
• In vitro crowding agents did not replicate the observed in-cell stabilization/destabilization trends.
• Cytoplasmic and nuclear lysates best mimicked in-cell folding, suggesting weak specific interactions are key.
• The unfolded state of barnase showed different stability and aggregation propensity in the cytoplasm versus the nucleus.
• Barnase folding in bacterial cells resembled nuclear folding but without aggregation at higher temperatures.

Conclusions:

• Protein folding and stability are significantly modulated by specific interactions within their native cellular environments.
• In situ studies are crucial for understanding and designing proteins that function accurately within cellular contexts.
• Cellular compartments and host organisms possess unique interaction landscapes that influence protein behavior.