Podolsky, K. A. | bioRxiv Science

bioRxiv2026

A peptide catalyst can replace an essential enzyme in a eukaryotic cell

Protein enzymes are central to modern biology, yet how catalysis emerged before the evolution of large, folded proteins remains unresolved. Here we show that a short, genetically encoded peptide can replace an essential enzyme in a living eukaryotic cell. We designed minimal peptides containing a Cys-Xaa-Cys catalytic motif and an endoplasmic reticulum retention signal, and identified variants that rescue the otherwise lethal deletion of protein disulfide isomerase (PDI) in Saccharomyces cerevisiae. Cells relying on these peptides remain viable, though they grow more slowly and adapt by activating stress-response pathways, consistent with PDI being replaced by catalysts of lower intrinsic efficiency. Biochemical analyses show that peptide activity depends on local chemical environment and secondary structure rather than a globular fold. These results demonstrate that short peptides can replace an essential cellular reaction in vivo at the system level, supporting the plausibility of peptide-based catalysis as a precursor to modern protein enzymes.

biochemistry↗

bioRxiv2021

Semisynthesis of functional transmembrane proteins in GUVs

Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

biochemistry↗

bioRxiv2020

Lipid Sponge Droplets as Programmable Synthetic Organelles

Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and non-ionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems. Significance statementOrganelles spatially and temporally orchestrate biochemical reactions in a cell to a degree of precision that is still unattainable in synthetic reaction systems. Additionally, organelles such as the endoplasmic reticulum (ER) contain highly interconnected and dense membrane networks that provide large reaction spaces for both transmembrane and soluble enzymes. We present lipid sponge droplets to emulate the functions of organelles such as the ER. We demonstrate that lipid sponge droplets can be programmed to internally concentrate specific proteins, host and accelerate biochemical transformations, and to rapidly and reversibly sequester and release proteins to control enzymatic reactions. The self-assembled and programmable nature of lipid sponge droplets will facilitate the integration of complex functions for bottom up synthetic biology.

synthetic biology↗