posted on 2020-03-17, 15:39authored bySüreyya
E. Geissinger, Andreas Schreiber, Matthias C. Huber, Lara G. Stühn, Stefan M. Schiller
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.