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

Area of Science:

• Biochemistry
• Cell Biology
• Protein Science

Background:

• In vitro protein studies often fail to replicate in-cell conditions.
• Small proteins are thought to be more affected by chemical interactions than macromolecular crowding within cells.

Purpose of the Study:

• To investigate how cellular environment, including intracellular compartments and host organisms, affects small protein folding.
• To compare in-cell protein behavior with in vitro models mimicking cellular conditions.

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 experiments using Ficoll (crowding) and M-PER™ (non-specific interactions), as well as cell lysates.

Main Results:

• Barnase folding was stabilized in the cytoplasm and destabilized in the nucleus of U2-OS cells.
• In vitro crowding and non-specific interaction mimics did not replicate cellular observations.
• Cellular lysates better mimicked in-cell barnase folding, suggesting weak specific interactions are key.
• Barnase exhibited an unstable, aggregation-prone unfolded state in the cytoplasm but a stable unfolded state in the nucleus.
• In bacterial cells, barnase folding resembled nuclear folding without aggregation at higher temperatures.

Conclusions:

• In-cell protein behavior is significantly influenced by specific weak interactions within cellular compartments.
• Protein interactions are adapted to their native cellular environments, emphasizing the need for in situ studies.
• Studying and designing proteins within their native cellular context (in situ) is crucial for accurate understanding and application.