Biology subjects

Morgner, N.

Publications and source records attributed to Morgner, N..

5 recordsLinked to original sources

bioRxiv2024

Dynamic basis of lipopolysaccharide export by LptB2FGC

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The {beta}-jellyroll domain of LptF stably interacts with the LptG and LptC {beta}-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF {beta}-jellyroll domain. In LptB2FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative en try site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its {beta}-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF {beta}-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.

biophysics↗

bioRxiv2024

The Mycobacterium lipid transporter MmpL3 is dimeric in detergent solution, SMALPs and reconstituted nanodiscs

The mycobacterial membrane protein large 3 (MmpL3) transports key precursor lipids to the outer membrane of Mycobacterium species. Multiple structures of MmpL3 from both M. tuberculosis and M. smegmatis in various conformational states indicate that the protein is both structurally and functionally monomeric. However, most other resistance, nodulation and cell division (RND) transporters structurally characterised to date are either dimeric or trimeric. Here we present an in depth biophysical and computational analysis revealing that MmpL3 from M. smegmatis exists as a dimer in a variety of membrane mimetic systems (SMALPs, detergent-based solution and nanodiscs). Sucrose gradient separation of MmpL3 populations from M. smegmatis, reconstituted into nanodiscs, identified monomeric and dimeric populations of the protein using laser induced liquid bead ion desorption (LILBID), a native mass spectrometry technique. Preliminary cryo-EM analysis confirmed that MmpL3 forms physiological dimers. Untargeted lipidomics experiments on membrane protein co-purified lipids revealed PE and PG lipid classes were predominant. Molecular dynamics simulations, in the presence of physiologically-relevant lipid compositions revealed the likely dimer interface.

biophysics↗

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↗