Biology subjects

Morgner, N.

Publications and source records attributed to Morgner, N..

3 recordsLinked to original sources

bioRxiv2023

Cell free expression in proteinosomes prepared from native protein-PNIPAAm conjugates

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalisation with an open, semi-permeable membrane. Critically, to date, there have been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, we present a novel approach to the fabrication of proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. We show that these native proteinosomes offer an excellent alternative as artificial cell chassis. Significantly, the native proteinosomes are stable under high salt conditions and can consequently support cell free transcription and translation. The native proteinosomes offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, we demonstrate the integration of proteinosomes into higher order cellular architectures with membrane free compartments and liposomes. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

synthetic biology↗

bioRxiv2023

Lateral gating mechanism and plasticity of the BAM complex in micelles and E. coli

The {beta}-barrel assembly machinery (BAM) mediates folding and insertion of the majority of OMPs in Gram-negative bacteria. BAM is a penta-heterooligomeric complex consisting of the central {beta}-barrel BamA and four interacting lipoproteins BamB, C, D, and E. The conformational switching of BamA between inward-open (IO) and lateral-open (LO) conformations is required for substrate recognition and folding. However, the mechanism for the lateral gating or how the structural details observed in vitro correspond with the cellular environment remains elusive. Here we addressed these questions by characterizing the conformational heterogeneity of BamAB, BamACDE and BamABCDE complexes in detergent micelles and or E. coli using pulsed dipolar electron spin resonance spectroscopy (PDS). We show that the binding of BamB does not induce any visible changes in BamA and the BamAB complex exists in the IO conformation. The BamCDE complex induces an IO to LO transition through a coordinated movement along the BamA barrel. However, the extracellular loop (L6) is unaffected by the presence of lipoproteins and exhibits a large segmental dynamics extending to the exit pore. PDS experiments with BamABCDE complex in intact E. coli confirmed the dynamic behavior of both the lateral gate and the L6 in the native environment. Our results demonstrate that the BamCDE complex plays a key role for the function by regulating lateral gating in BamA.

biophysics↗

bioRxiv2023

Two conformations of the Tom20 preprotein receptor in the TOM holo complex

The TOM complex is the main entry point for precursor proteins into mitochondria. Precursor proteins containing targeting sequences are recognized by the TOM complex and imported into the mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle cryoEM at 3.3 [A] resolution, showing its interaction with a bound presequence at 4 [A] resolution, and of the TOM holo complex including the Tom20 receptor at 6-7 [A] resolution. TOM is a transmembrane complex consisting of two {beta}-barrels, three receptor subunits and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding precursor proteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.

biophysics↗