Adjustable Bioorthogonal Conjugation Platform for Protein Studies in Live Cells Based on Artificial Compartments

Area of Science:

• Synthetic biology and bioconjugation chemistry.
• In vivo protein scaffolding and molecular assembly.

Background:

• Investigating complex biological processes in vivo requires precise spatial organization of functional molecules on 3D structures.
• Existing in vivo scaffolding methods often lack covalent conjugation on confined scaffolds and precise quantitative characterization.

Purpose of the Study:

• To develop a bioorthogonal dual conjugation platform for in vivo scaffolding.
• To enable precise stoichiometric quantification of conjugated entities on artificial compartments.

Main Methods:

• Implementation of SpyTag/SpyCatcher (ST/SC) and strain-promoted azide-alkyne cycloaddition (SPAAC) bioorthogonal reactions.
• Utilized self-assembled protein membrane-based compartments (PMBCs) as scaffolds for in vivo conjugation.
• Quantified reaction yields and absolute concentrations using tandem mass spectrometry.

Main Results:

• Achieved a SPAAC reaction yield of 23% ± 3% and ST/SC surface conjugation yield of 82% ± 9%.
• Demonstrated compatibility of both conjugation chemistries and enhanced proteolytic stability of the compartments.
• Quantified absolute concentrations of tethered proteins (mCherry-ST-His and pAzF-SC-E20F20-His) in attomole/cell range.

Conclusions:

• The established in vivo conjugation platform allows for quantifiable protein-protein interaction studies on geometrically defined scaffolds.
• This platform facilitates future investigations into the effects of scaffold-tethering on enzyme activity in vivo.