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

Abstract

The investigation of complex biological processes in vivo often requires defined multiple bioconjugation and positioning of functional entities on 3D structures. Prominent examples include spatially defined protein complexes in nature, facilitating efficient biocatalysis of multistep reactions. Mimicking natural strategies, synthetic scaffolds should comprise bioorthogonal conjugation reactions and allow for absolute stoichiometric quantification as well as facile scalability through scaffold reproduction. Existing in vivo scaffolding strategies often lack covalent conjugations on geometrically confined scaffolds or precise quantitative characterization. Addressing these shortcomings, we present a bioorthogonal dual conjugation platform based on genetically encoded artificial compartments in vivo, comprising two distinct genetically encoded covalent conjugation reactions and their precise stoichiometric quantification. The SpyTag/SpyCatcher (ST/SC) bioconjugation and the controllable strain-promoted azide−alkyne cycloaddition (SPAAC) were implemented on self-assembled protein membrane-based compartments (PMBCs). The SPAAC reaction yield was quantified to be 23% ± 3% and a ST/SC surface conjugation yield of 82% ± 9% was observed, while verifying the compatibility of both chemical reactions as well as enhanced proteolytic stability. Using tandem mass spectrometry, absolute concentrations of the proteinaceous reactants were calculated to be 0.11 ± 0.05 attomol/cell for PMBC surface-tethered mCherry-ST-His and 0.22 ± 0.09 attomol/cell for PMBC-constituting pAzF-SC-E20F20-His. The established in vivo conjugation platform enables quantifiable protein–protein interaction studies on geometrically defined scaffolds and paves the road to investigate effects of scaffold-tethering on enzyme activity.

To View Your Biomolecule, Click inside the Cell

“…[79] Although SPAACp erforms less efficiently than IEDDA, it has nevertheless been shown to be applicable for intracellular purposes as well despite sluggish kinetics. [80,81] For SPAACl abelling of azido-UAAs,l ipophilicity is ac rucial criterion in the choice of cyclooctyne for solubility,c ellpermeability,and aspecificity issues.The use of dibenzoannulated probes such as DIBAC/DBCO is challenging for these reasons,w hile the less hydrophobic BCN derivatives react more slowly with alkyl azides than with aryl azides. [32] BCN can, however,b eavaluable alternative even for aliphatic Scheme 5.…”

To View Your Biomolecule, Click inside the Cell

“…Although SPAAC performs less efficiently than IEDDA, it has nevertheless been shown to be applicable for intracellular purposes as well despite sluggish kinetics [80, 81] . For SPAAC labelling of azido‐UAAs, lipophilicity is a crucial criterion in the choice of cyclooctyne for solubility, cell‐permeability, and aspecificity issues.…”

“…[79] Although SPAACp erforms less efficiently than IEDDA, it has nevertheless been shown to be applicable for intracellular purposes as well despite sluggish kinetics. [80,81] For SPAACl abelling of azido-UAAs,l ipophilicity is ac rucial criterion in the choice of cyclooctyne for solubility,c ellpermeability,and aspecificity issues.The use of dibenzoannulated probes such as DIBAC/DBCO is challenging for these reasons,w hile the less hydrophobic BCN derivatives react more slowly with alkyl azides than with aryl azides. [32] BCN can, however,b eavaluable alternative even for aliphatic azides,asrecently illustrated by Tirrell and Ho in bacteria.…”

Intracellular Assembly of Interacting Enzymes Yields Highly‐Active Nanoparticles for Flow Biocatalysis

“…[11,13] Since our AEH design can be readily applied to multiple classes of enzymes, we wanted to explore the possibilities of producing these materials in living cells for use in diverse flow biocatalysis formats. Previous examples of SC/ST-mediated enzyme conjugation in living bacteria were limited to coexpression of bacterial microcompartment shells, [14] compartment-forming mediators such as elastin-like protein [15] or the crystallizing Cry protein, [16] or resulted in uncontrolled formation of inclusion bodies that must be further stabilized by chemical cross-linking. [17] Therefore, to enable independently controllable bifunctional enzyme aggregation in E. coli, in this work polycistronic vectors were cloned containing genes encoding for a (R)-stereoselective alcohol dehydrogenase (ADH) and a glucose-1-dehydrogenase (GDH) fused with SC and ST domains, respectively (Figure 1).…”