Biology subjects

Abdelkader, E. H.

Publications and source records attributed to Abdelkader, E. H..

3 recordsLinked to original sources

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↗