Biology subjects

Di Trani, J. M.

Publications and source records attributed to Di Trani, J. M..

5 recordsLinked to original sources

bioRxiv2024

Cryo-EM of native membranes reveals an intimate connection between the Krebs cycle and respiration in mycobacteria

Imaging of endogenous protein complexes in their native membranes can reveal protein-protein interactions that are lost upon detergent solubilization. To investigate interactions in the mycobacterial oxidative phosphorylation machinery, we prepared inverted membrane vesicles from Mycobacterium smegmatis and enriched for vesicles containing complexes of interest by affinity chromatography. Electron cryomicroscopy (cryo-EM) of these vesicles revealed that malate-quinone oxidoreductase (Mqo), an enzyme from the Krebs cycle, physically associates with the electron transport chain Complex III2IV2 (CIII2CIV2) supercomplex. Analysis of the Mqo:CIII2CIV2 interaction shows that CIII2CIV2 is necessary for malate-driven, but not NADH- driven, electron transport chain activity and oxygen consumption. Further, the association of Mqo with CIII2CIV2 enables electron transfer from malate to CIII2CIV2 with millisecond kinetics. Together, these findings indicate a connection between the Krebs cycle and respiration that directs electrons along a single branch of the mycobacterial electron transport chain.

biochemistry↗

bioRxiv2024

High resolution cryo-EM of V-ATPase in native synaptic vesicles

Neurotransmitter release into the synaptic cleft between neurons enables the intercellular communication central to the function of the brain. In the presynaptic neuron, the proton pumping vesicular- or vacuolar-type ATPase (V-ATPase) powers neurotransmitter loading into synaptic vesicles (SVs), with the V1 complex dissociating from the membrane region of the enzyme before exocytosis. We isolated SVs from rat brain using SidK, a V-ATPase-binding bacterial effector protein. Single particle electron cryomicroscopy of the vesicles allowed high-resolution structure determination of V-ATPase within the native SV membrane. In the structure, regularly spaced cholesterol molecules decorate the enzymes rotor and the abundant SV protein synaptophysin binds the complex stoichiometrically. Conditions where ATP hydrolysis drives glutamate loading result in loss of V1 from the SV membrane, suggesting that SV loading is sufficient to induce V-ATPase dissociation.

biochemistry↗

bioRxiv2022

Flexible client-dependent cages in the assembly landscape of the periplasmic protease-chaperone DegP

The periplasmic protein DegP, that is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that maintains protein homeostasis in gram-negative bacteria. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo-oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming cage ensembles that can include extremely large cage particles. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of flexible cage structures including novel 30mer and 60mer particles. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size. Significance statementGram-negative pathogens export virulence factors that interfere with the function of host cells. This process is mediated by DegP, a protein which controls protein homeostasis in the periplasm of these bacteria and thus is a target for the development of novel antibiotics. DegP operates by incorporating client proteins inside cage-like structures to either recycle them or protect them from aggregation. Using a combination of dynamic light scattering measurements and cryogenic electron microscopy, we have shown that DegP can adopt many types of cages, some as large as subcellular organelles, depending on the size of the engaged client. This property likely enables DegP to capture different sized clients in response to protein misfolding stresses.

biophysics↗

bioRxiv2021

Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203)

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than fifty years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.

biochemistry↗

bioRxiv2019

Shake-it-off: A simple ultrasonic cryo-EM specimen preparation device

Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM specimen preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments.\n\nSynopsisA method is presented for high-speed low-volume cryo-EM specimen preparation with a device constructed from readily available components.

biophysics↗