Biology subjects

Ribezzi-Crivellari, M.

Publications and source records attributed to Ribezzi-Crivellari, M..

4 recordsLinked to original sources

bioRxiv2025

Design and experimental characterization of specificity-switching mutational paths of WW domains

Specific interactions between proteins and other biomolecules are ubiquitous in cellular processes.How specificity is encoded in the protein sequence and can be modified through a minimal set of concerted mutations is a complex issue. In this work, we focus on the WW protein domain, whose variants specifically bind to different classes of proline-rich peptides. Combining unsupervised learning of homologous WW sequence data with Restricted Boltzmann Machines (RBM) and path-sampling methods, we design mutational paths of putative WW domains interpolating between two natural WW domains with either distinct or similar specificities. Sequences along the designed paths are then experimentally validated with high-throughput in-vitro binding assays against 3 peptides of different classes. The vast majority (93%) of intermediate sequences along the designed paths are responsive to the initial or/and final peptides. On the contrary, domains along scrambled paths, in which the same mutations are introduced in random order are not functional, emphasizing how successful design crucially depends on the ability to model epistatic interactions. Switch in specificity between classes I and IV, whose representative peptides bind to different pockets on the WW domain takes place through intermediates displaying some level of binding cross-reactivity with the tested peptides, contrary to the transition from class I to II, which are associated with the same binding pocket. Lastly, we show that the RBM paths share a high identity with internal nodes obtained from ancestral sequence reconstruction based on the seed WW domains.

bioengineering↗

bioRxiv2025

DNA barcoding for parallelised single-molecule characterisation of kinetic phenotypes

Single-molecule techniques provide exceptional resolution of biomolecular dynamics but are limited by low-throughput. We present a barcoding strategy that enables simultaneous kinetic profiling of multiple protein variants at the single-molecule level. Each protein is covalently linked to a unique DNA barcode, decoded via transient hybridisation of fluorescent probes distinguished by colour and binding duration. We applied this method to a library of 16 ClpS variants to systematically explore evolutionary trajectories towards a variant adapted to recognise N-terminal amino acids with binding kinetics optimised for single-molecule peptide sequencing. The approach uncovered [~]5-fold variation in median dissociation rates across variants. Several variants displayed bimodal kinetics, likely reflecting structural subpopulations, and differences in kinetic properties arose primarily from the relative abundance of these phenotypes. This approach offers a general framework for screening and optimising proteins for single-molecule applications, while revealing mechanistic insights that are inaccessible to traditional ensemble methods.

biophysics↗

bioRxiv2025

Parallelization of single-molecule binding kinetic measurements via protein barcode sequencing

Screening protein variants for desired functions has long relied on coupling of genotype (gene sequence) to phenotype (protein function), limiting the use of powerful single-molecule (SM) techniques. Here, we introduce a scalable SM screening method that bypasses this constraint by linking SM functional analysis to protein identity through SM protein sequencing. Protein variants are tagged with unique C-terminal peptide barcodes and loaded onto a semiconductor chip containing millions of nanowells. Protein-ligand interactions are monitored in real time at the SM level, and a dye-cycling strategy extends the measurable dynamic range, enabling quantification of slow dissociation rates typical of high-affinity interactions. After functional analysis, each protein molecule is identified by sequencing its barcode. We apply this method to 20 barcoded nanobodies spanning over 1,000-fold in affinity, yielding results consistent with published values and individual SM measurements. Our approach should accelerate protein engineering by enabling rapid, multiplexed SM screening of protein libraries.

biochemistry↗

bioRxiv2024

Development of a generalisable tryptophan-optimised quenchbody biosensor based on a synthetic nanobody library

Quenchbodies, antibodies labelled with fluorophores that increase in intensity upon antigen binding, offer great promise for biosensor development. Nanobody-based quenchbodies are particularly attractive due to their small size, ease of expression, high stability, rapid evolvability, and amenability to protein engineering. However, existing designs for protein detection show limited dynamic range, with fluorescence increases of only 1.1-1.7 fold. Here we identify the tryptophan residues in the nanobody complementarity-determining regions (CDRs) that are critical to quenchbody performance. Using a combination of rational design and molecular dynamics simulations, we developed an optimised nanobody scaffold with tryptophans introduced at key positions. We used this scaffold in an in vitro directed-evolution screen against human inflammatory cytokine interleukin-6 (IL-6). This yielded quenchbodies with 1.5-2.4-fold fluorescence increases, enabling IL-6 detection down to 2 nM. Our scaffold provides a valuable platform for developing biosensors for diverse protein targets, with applications in research, diagnostics, and environmental monitoring.

bioengineering↗