Biology subjects

Otting, G.

Publications and source records attributed to Otting, G..

7 recordsLinked to original sources

bioRxiv2026

Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes

Quantitative fluorescence lifetime imaging microscopy (FLIM) is limited by a lack of probes that combine single-residue specificity, minimal steric perturbation, visible-light excitation, and strong environment-dependent lifetime contrast. We introduce benzodiazole-based non-canonical amino acids (BD-ncAAs) as compact, genetically encoded sensors for site-specific FLIM. Using an engineered pyrrolysyl-tRNA synthetase pair, we incorporate three different BD-ncAAs into proteins in living cells. Site-specific incorporation of BD-ncAAs into a nanobody at different positions preserved native structure and antigen binding. The lead probe, cyano-substituted CBDS, exhibits visible-light excitation, large Stokes shifts, and pronounced environment-dependent fluorescence lifetimes spanning 1 to 24 ns. Under wash-free, live-cell FLIM conditions, CBDS quantitatively resolves localized structural microenvironments and transmembrane hydration gradients, while also distinguishing distinct states during protein secretion in eukaryotes. BD-ncAAs present genetically encoded, minimally perturbing reporters for tracking target proteins, detecting interactions with binding partners, and quantifying site-specific solvent accessibility in living cells by quantitative FLIM.

biophysics↗

bioRxiv2026

Isosteric Engineering of Enzymes: Overcoming Activity-Stability Trade-offs by Site-Selective CH -> N Substitutions

Enzymes used on industrial scale are routinely engineered for best performance. However, exhaustive mutagenesis campaigns using the twenty canonical proteinogenic amino acids rapidly reach an evolutionary ceiling, where gains in activity compromise other critical properties such as thermal endurance. Although non-canonical amino acids (ncAA) expand the chemical space, most are costly for use on an industrial scale and significantly perturb structure. Here, we demonstrate that the evolutionary ceiling of highly optimized polyethylene terephthalate (PET) hydrolases (PETases) can be broken with azatryptophans that (i) differ minimally from their canonical tryptophan, (ii) are genetically encoded, and (iii) are produced in high yield by enzymatic biosynthesis from inexpensive precursors. The first genetic encoding systems are described for 4-azatryptophan, 5-azatryptophan, and 6-azatryptophan, achieving single, site-selective isosteric CH [->] N substitutions that enhancing the catalytic activity while preserving thermal stability. The fluorescence of 6AW provides a uniquely sensitive reporter of side-chain solvent exposure, which is critical for PETase activity and shown to vary between five different PETases. Furthermore, Azatryptophan-bearing enzymes are inexpensive to produce. To benchmark PETase activity, a rapid fluorescence-based kinetic assay, PETra, is introduced, which delivers consistency and reproducibility by using a soluble substrate yet correlates strongly with the hydrolysis of solid PET.

biochemistry↗

bioRxiv2025

Biosynthetic lanthanide-luminescent mini-proteins using genetic code expansion

Non-canonical amino acids (ncAA) are promising as light-harvesting antennae for lanthanide luminescence in lanthanide-binding peptides and proteins. Here we present empirical insights into antenna-lanthanide interactions which reveal design principles of bright luminescent proteins. Peptides designed to act as lanthanide binding tags (LBT) show a trade-off between sensitization and lanthanide binding affinity. We generated a new protein, termed RF2, through computational design with nano-molar binding affinity and more than two-fold increase in terbium(III) luminescence. In this scaffold, 6-azatryptophan (6AW) achieved a ten-fold enhancement of the europium(III) luminescence in vivo. The RF2 6AW mutant also sensitizes the luminescence of dysprosium(III) and samarium(III). These results demonstrate the capability of de novo protein design to produce highly luminescent lanthanide-binding mini-proteins with a genetically encoded ncAA antenna.

biophysics↗

bioRxiv2024

Rendering Proteins Fluorescent Inconspicuously: Genetically Encoded 4-Cyanotryptophan Conserves Their Structure and Enables the Detection of Ligand Binding Sites

Cyano-tryptophans (CN-Trp) are privileged multimodal reporters on protein structure. They are similar in size to the canonical amino acid tryptophan and some of them exhibit bright fluorescence which responds sensitively to changes in the environment. We selected aminoacyl-tRNA synthetases specific for 4-, 5-, 6-, and 7-CN-Trp for high-yield in vivo production of proteins with a single, site-specifically introduced nitrile label. The absorption maximum of 4-CN-Trp is distinct from Trp, allowing the selective excitation of its intense fluorescence. 4-CN-Trp features bright fluorescence in the visible range. Crystal structures of maltose binding protein demonstrate near-complete structural conservation when a native buried Trp residue is replaced by 4-CN-Trp. Besides presenting an inconspicuous tag for live cell microscopy, the high fluorescence of 4-CN-Trp enables measurements of subnanomolar ligand binding affinities in isotropic solution, as demonstrated by the complex between rapamycin and the peptidyl-prolyl isomerase FKBP12 furnished with a 4-CN-Trp residue in the substrate binding pocket. Furthermore, 4-CN-Trp residues positioned at different locations of a protein containing multiple tryptophan residues permits using fluorescence quenching experiments to detect the proximity of individual Trp residues to the binding site of aromatic ligands.

biochemistry↗

bioRxiv2021

Main protease mutants of SARS-CoV-2 variants remain susceptible to PF-07321332

The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic nirmatrelvir (PF-07321332). We expressed Mpro of six SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, B.1.1.529 Omicron, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, P132H, L205V). Enzyme kinetics showed that these Mpro variants are similarly catalytically competent as the wildtype. We show that nirmatrelvir has similar potency against the variants as against the wildtype. Our in vitro data suggest that the efficacy of the specific Mpro inhibitor nirmatrelvir is not compromised in current COVID-19 variants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/470226v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@182cc6org.highwire.dtl.DTLVardef@1239d10org.highwire.dtl.DTLVardef@11ca6d9org.highwire.dtl.DTLVardef@df53b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

biochemistry↗

bioRxiv2020

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including double electron-electron resonance (DEER), on reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.

biochemistry↗