The extracellular protein VlsE is destabilized inside cells

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Cellular environments present unique conditions affecting protein stability and folding.
• Understanding intracellular protein behavior is crucial for cell biology and disease research.

Purpose of the Study:

• To investigate how the intracellular environment differentially impacts the stability and folding kinetics of two distinct proteins: PGK and VlsE.
• To explore the mechanisms behind these observed effects using advanced imaging techniques.

Main Methods:

• Utilized U2OS cells as in vivo models.
• Employed fast relaxation imaging, combining temperature jumps with Förster resonance energy transfer (FRET) microscopy.
• Analyzed protein folding stability and kinetics in both cellular and aqueous buffer conditions.

Main Results:

• The cytoplasmic enzyme phosphoglycerate kinase (PGK) exhibited increased stability within cells.
• The cell surface antigen VlsE showed decreased stability and slower folding kinetics inside cells compared to buffer.
• FRET measurements indicated VlsE adopts a more compact state within cells, distinct from its native aqueous buffer conformation.
• Confirmed VlsE stabilization by the crowding agent Ficoll in vitro, contrasting its destabilization in vivo.

Conclusions:

• The intracellular environment has opposing effects on the stability of different proteins based on their evolutionary context and structure.
• Proposed mechanisms for VlsE destabilization include competing long-range interactions or shape-dependent crowding effects within the cell.
• Highlighted the complexity of protein folding and stability within the crowded cellular milieu.