Biology subjects

Filius, M.

Publications and source records attributed to Filius, M..

7 recordsLinked to original sources

bioRxiv2025

Generative Design of High-Affinity Peptides Using BindCraft

Discovering high-affinity ligands directly from protein structures remains a key challenge in drug discovery. We applied BindCraft, a structure-guided generative modeling platform, to de novo design of peptide ligands for protein interfaces. Originally developed for miniprotein binders, we evaluated its use for shorter peptides (10-20mers) as peptides hold greater synthetic accessibility and therapeutic potential. For the oncoprotein MDM2, BindCraft generated 70 unique peptides; 15 were synthesized, and 7 showed specific binding with nanomolar affinities (KD = 65-165 nM). Competition assays confirmed site-specific binding for the intended target site. For another oncology target, WDR5, peptides were designed for the MLL (WIN) and MYC (WBM) sites. Of the peptides tested for each site, no validated hits were found for the WIN site, but six candidates bound the WBM site with sub-micromolar affinity (KD = 219-650 nM). Based on Bindcrafts structural prediction of the binding interface, we designed a stapled variant of the best WDR5 binder, improving the potency by 6-fold to a KD of 39 nM. Overall, our findings establish BindCraft as a powerful and accessible platform for structure-based peptide discovery, with remaining limitations, but with a promising success rate even for challenging targets.

biochemistry↗

bioRxiv2023

Full-Length Single-Molecule Protein Fingerprinting

Proteins are the primary functional actors of the cell. Hence, their identification is pivotal to advance our understanding of cell biology and disease. Current protein analysis methods are of limited use for distinguishing proteoforms. In particular, mass spectrometric methods often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate FRET-based single-molecule protein fingerprinting to map the location of individual amino acids and a post-translational modification within single full-length protein molecules. Using an approach that relies on transient binding of fluorescently labeled DNA strands to probe the amino acids on a protein one by one we show that we can fingerprint intrinsically disordered proteins as well as folded globular proteins with sub-nanometer resolution. We anticipate that this technology will be used for proteoform identification in biological and translational research with ultimate sensitivity.

biophysics↗

bioRxiv2021

Evaluation of FRET X for Single-Molecule Protein Fingerprinting

Single-molecule protein identification is a novel, as of yet unrealized concept with potentially groundbreaking applications in biological research. We propose a method called FRET X (Forster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands, and accumulated FRET efficiency values constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on a system of model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics.

biophysics↗

bioRxiv2020

High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object. The limited number of available FRET pair fluorophores and complicated data analysis makes it challenging to apply single-molecule FRET for structural analysis of biomolecules. Currently, only a couple of FRET pairs can be reliably measured on a single object. Here we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the FRET efficiency and pair distance with sub-nanometer resolution. We determine the distance between other pairs by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. We envision that our FRET X technology will be a tool for the high-resolution structural analysis of biomolecules and other nano-structures.

biophysics↗

bioRxiv2020

FRETboard: semi-supervised classification of FRET traces

AO_SCPLOWBSTRACTC_SCPLOWForster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nano-scale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner, but their operating system-dependent installation requirements and lack of flexibility impede wide-spread adoption. Here we propose to fit such models more efficiently and intuitively by adopting a semi-supervised approach, in which the user interactively guides the model to fit a given dataset, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios, and correctly estimate parameters for up to eleven states. On in vitro data we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis. Availabilitysource code is available at https://github.com/cvdelannoy/FRETboard. The FRETboard classification tool is also available as a browser application at https://www.bioinformatics.nl/FRETboard.

biophysics↗

bioRxiv2020

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT

Super-resolution imaging allows for visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA Point Accumulation In Nanoscale Topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), that allows for faster binding. Ago pre-orders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude while maintaining the high spatial resolution. We envision this tool to be useful not only for super-resolution imaging, but also for other techniques that rely on nucleic-acid interactions.

biophysics↗

bioRxiv2017

Single-Molecule Peptide Fingerprinting

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study, such as single-cell analysis, challenging. Here we demonstrate a proof of concept of singlemolecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits uni-directional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.\n\nSIGNIFICANCEProtein sequencing remains a challenge for small samples. A sensitive sequencing technology will create the opportunity for single-cell proteomics and real-time screening for on-site medical diagnostics. In order to resolve protein identity, we previously developed a computational algorithm that analyzes the ordered sequence of only two types of amino acids within a protein species. Through modification of a biological nanomachine, here we developed single-molecule fluorescence technology to linearize protein molecules and to read signals from labeled amino acids in an ordered manner. This proof of concept of singlemolecule fingerprinting will open a new door to single-molecule protein sequencing and pave the road towards the development of a new, fast, and reliable diagnostic tool.

biophysics↗