Protein charge determination and implications for interactions in cell extracts

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Biophysics

Background:

• Protein charge is crucial for stability and solubility in dilute solutions.
• Understanding protein charge under physiological conditions is essential for native behavior.
• Cytochrome c is a key protein for studying charge effects in vivo.

Purpose of the Study:

• To investigate the charge of cytochrome c in physiological solutions, specifically in buffer and Escherichia coli extracts.
• To determine how factors like sulfate binding and macroanion interactions affect protein charge.
• To analyze the impact of charge modifications on protein behavior and interactions within cellular environments.

Main Methods:

• Utilized free boundary electrophoresis to measure protein charge.
• Employed native gel electrophoresis for characterization in complex cellular extracts.
• Created and analyzed cytochrome c mutants with altered charge properties.

Main Results:

• Cytochrome c charge was approximately two-fold lower than predicted from its primary structure.
• Sulfate binding and interactions with macroanions in E. coli extracts significantly tuned cytochrome c charge, rendering it anionic.
• Mutants with reduced cationic residues became charge-neutral and exhibited inert behavior in extracts.
• Negative charge was found to be critical for stabilizing physiological environments and preventing aggregation via charge-charge repulsion and preferential hydration.

Conclusions:

• Protein charge in physiological environments deviates significantly from predictions based on primary structure alone.
• Interactions with cellular components, such as macroanions and ions like sulfate, play a vital role in modulating protein charge.
• Engineered charge reduction can lead to "inert" protein behavior, highlighting the importance of net charge in preventing aggregation and maintaining molecular organization in vivo.
• The findings emphasize the role of negative charge and charge-charge repulsion in maintaining protein solubility and preventing aggregation within the crowded cellular milieu.