The Effect of Protein Tagging on Aggregation and Phase Separation

Area of Science:

• Biochemistry and Molecular Biology
• Protein Biophysics
• Structural Biology

Background:

• Protein tags are essential tools for protein manipulation, including purification, solubilization, detection, and imaging.
• However, these exogenous elements can interfere with the intrinsic self-assembly properties of proteins, especially intrinsically disordered proteins and low-complexity domains.

Purpose of the Study:

• To review how various protein tags (affinity, solubility, fluorescent, chemical) influence protein aggregation, amyloid formation, and liquid-liquid phase separation (LLPS).
• To classify the mechanisms by which tags perturb protein self-assembly.
• To provide strategies for distinguishing intrinsic protein behavior from tag-induced artifacts.

Main Methods:

• Literature review and classification of perturbation mechanisms.
• Analysis of case studies across diverse protein systems (pathogenic amyloids, RNA-binding proteins, prions, etc.).
• Discussion of experimental strategies for validation and interpretation.

Main Results:

• Identified six primary categories of tag perturbation mechanisms: solubility enhancement, artificial multivalency, electrostatic interactions, local effects, metal coordination, and positional dependence.
• Demonstrated that tags significantly alter assembly kinetics, phase boundaries, material properties, fibril morphology, oligomeric states, and observed phenotypes.
• Observed that tags can either suppress or promote protein self-assembly, leading to non-native condensations or stabilization of alternative aggregate states.

Conclusions:

• Protein tags are not inert tools but active variables that profoundly shape protein self-assembly states.
• Experimental design must account for tag-induced effects to accurately study intrinsic protein behavior.
• Careful interpretation of results and orthogonal validation methods are crucial for distinguishing intrinsic versus construct-dependent phenomena.