CIISB Research Highlights Archive

Detection of SARS-CoV-2 N protein. (A) UCNP label: Alkyne-PEG-neridronate strongly binds via two phosphonate groups to surface lanthanide ions of UCNPs, and a click reaction binds the conjugate to azide-modified streptavidin. (B) Scheme of sandwich ULISA: A microtiter plate is coated with two monoclonal antibodies that capture the N protein. Then, two biotinylated detection antibodies bind to the N protein. The sandwich immune complex is finally detected by using the UCNP label.

Hans H. Gorris Research Group

Significance

The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.

Brandmeier, J.C., Jurga, N., Grzyb, T., Hlaváček, A., Obořilová, R., Skládal, P., Farka, Z., and Gorris, H.H.: Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay, Anal. Chem. 2023 95, 51, 4753 – 4759, https://doi.org/10.1021/acs.analchem.2c05670

This study experimentally determines the properties that would have accompanied some of the most feasible ncAA candidates from the prebiotic pool. We incorporated the selected ncAAs into combinatorial peptide libraries along with (or replacing) other early amino acids to evaluate their effect on fundamental physicochemical properties such as solubility and secondary structure formation.

Stephen D. Fried and Klara Hlouchová Research Groups

Significance

Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 “early” AAs and that the 10 “late” AAs were products of biosynthetic pathways. However, many nonproteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet, and would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluate the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces. The results show that foldability was likely a critical factor in the selection of the canonical alphabet. Unbranched aliphatic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are oversolubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides’ secondary structure potential, for which we suggest a biophysical model. Our results support the view that, despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of protein evolution.

Makarov M. et al.: Early Selection of the Amino Acid Alphabet Was Adaptively Shaped by Biophysical Constraints of Foldability, J. Amer. Chem. Soc. (2023), 145, 5320-5329, https://doi.org/10.1021/jacs.2c12987

Overview of the dormant ribosome structure from 1 hpf zebrafish (a) and Xenopus egg (b). Ribosome-associated factors are shown as surface representations; eEF2b and eIF5a correspond to Protein Data Bank (PDB) accessions 6MTE and 5DAT, respectively.

Andrea Pauli Research Group

Significance

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4–eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

Leesch, F. et. al.: A molecular network of conserved factors keeps ribosomes dormant in the egg, Nature (2023), 613, 712-720: https://doi.org/10.1038/s41586-022-05623-y

Interaction of Myxovalargin with the E. coli ribosome. (a) Relative position of P-site tRNA (green) and erythromycin (Ery, cyan) to 23S rRNA nucleotides protected from DMS by 10 μM (light blue) or 100 μM (dark blue) MyxB.

Andreas Kirschning, Daniel N. Wilson, and R. Müller Research Groups

Significance

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Koller, T.O., Scheid, U., et al.: The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition, J. Amer. Chem. Soc. (2023), 145, 851-863 https://doi.org/10.1021/jacs.2c08816

Features comparison between T4P-like structure obtained by cryo-EC, remote homology detection, and subtomogram averaging. The main dimensions of the T4P-like complex obtained by 3D cryo-EC (Center Left; EMD-14097), remote homology detection (Center), and subtomogram averaging (Center Right; EMD-14096) are compared. The Left and Right boxes show comparable images of the slice through at equivalent levels, from Top to Bottom, for the T4P-like complex obtained by cryo-EC (left box) and by cryo-ET subtomogram averaging (right box), both computed imposing a p6 symmetry. The dark-yellow boxes indicate the noncrystalline regions that are missing in the model obtained by cryo-EC with respect to the model obtained by subtomogram averaging. The S-layer (SL), the outer membrane (OM), and the inner membrane (IM) thicknesses are indicated. Scale bars indicate 50 Å.

Dario Piano Research Group

Significance

The cell envelope of the extremophile bacterium Deinococcus radiodurans was studied by cryo-electron microscopy and described with unprecedented detail. In this bacterium, the outermost cell envelope layer, named surface layer, is characterized by a highly regular tiling of proteins extending their crystalline organization to the cell envelope layers below (until the inner membrane). The study shows three main protein complexes, with masses in the MDa range, regularly organized into an astonishing geometrical regularity. The observed organization contributes to protecting the cell against environmental stressors and maintaining an efficient permeation of environmental solutes.

Surface layers (S-layers) are highly ordered coats of proteins localized on the cell surface of many bacterial species. In these structures, one or more proteins form elementary units that self-assemble into a crystalline monolayer tiling the entire cell surface. Here, the cell envelope of the radiation-resistant bacterium Deinococcus radiodurans was studied by cryo-electron microscopy, finding the crystalline regularity of the S-layer extended into the layers below (outer membrane, periplasm, and inner membrane). The cell envelope appears to be highly packed and resulting from a three-dimensional crystalline distribution of protein complexes organized in close continuity yet allowing a certain degree of free space. The presented results suggest how S-layers, at least in some species, are mesoscale assemblies behaving as structural and functional scaffolds essential for the entire cell envelope.

Farci, D., Haniewicz, P., and Piano, D.: The structured organization of Deinococcus radiodurans’ cell envelope, PNAS (2022) e112101, https://doi.org/10.1073/pnas.2209111119

Posttranslational modifications of tubulin are expected to control the functions of a wide range of microtubule-interacting proteins. Here, in vitro reconstitution assays with purified brain tubulin from mouse models lacking specific tubulin-modifying enzymes show that tubulin modification patterns selectively impact microtubule-protein interactions.

Carsten Janke and Marcus Braun Research Group

Significance
Tubulin posttranslational modifications have been predicted to control cytoskeletal functions by coordinating the molecular interactions between microtubules and their associating proteins. A prominent tubulin modification in neurons is polyglutamylation, the deregulation of which causes neurodegeneration. Yet, the underlying molecular mechanisms have remained elusive. Here, using in-vitro reconstitution, we determine how polyglutamylation generated by the two predominant neuronal polyglutamylases, TTLL1 and TTLL7, specifically modulates the activities of three major microtubule interactors: the microtubule-associated protein Tau, the microtubule-severing enzyme katanin and the molecular motor kinesin-1. We demonstrate that the unique modification patterns generated by TTLL1 and TTLL7 differentially impact those three effector proteins, thus allowing for their selective regulation. Given that our experiments were performed with brain tubulin from mouse models in which physiological levels and patterns of polyglutamylation were altered by the genetic knockout of the main modifying enzymes, our quantitative measurements provide direct mechanistic insight into how polyglutamylation could selectively control microtubule interactions in neurons.

Genova, M., Grycova, L., Puttrich, V., Magiera, M.M., Lansky, Z., Janke, C., and Braun, M.:

Tubulin polyglutamylation differentially regulates microtubule-interacting proteins

EMBO Journal, (2023) e112101, https://doi.org/10.15252/embj.2022112101