Rapid Assessment of Size, Shape, and Chemical Complementarity of Ligands for Computational Protein Design

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Chubb , View ORCID Profile Andrey V. Romanyuk , View ORCID Profile Dominic Alibhai , View ORCID Profile Jennifer J. McManus , View ORCID Profile Graham J. Leggett , View ORCID Profile Nigel S. Scrutton , View ORCID Profile Thomas A. A. Oliver , View ORCID Profile Derek N. Woolfson doi: https://doi.org/10.1101/2025.06.30.662286 Rokas Petrenas 1 School of Chemistry, University of Bristol, Bristol, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rokas Petrenas Katarzyna Ozga 1 School of Chemistry, University of Bristol, Bristol, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Katarzyna Ozga Joel J. Chubb 1 School of Chemistry, University of Bristol, Bristol, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Joel J. Chubb Andrey V. Romanyuk 2 Max Planck-Bristol Centre for Minimal Biology, University of Bristol, Bristol, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrey V. Romanyuk Dominic Alibhai 3 Wolfson Bioimaging Facility, University of Bristol, Bristol, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dominic Alibhai Jennifer J. McManus 4 HH Wills Physics Laboratory, University of Bristol, Bristol, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jennifer J. McManus Graham J. Leggett 5 Department of Chemistry, University of Sheffield, Sheffield, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Graham J. Leggett Nigel S. Scrutton 6 Manchester Institute of Biotechnology, University of Manchester, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nigel S. Scrutton Thomas A. A. Oliver 1 School of Chemistry, University of Bristol, Bristol, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas A. A. Oliver Derek N. Woolfson 1 School of Chemistry, University of Bristol, Bristol, UK; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Derek N. Woolfson For correspondence: d.n.woolfson{at}bristol.ac.uk Abstract Info/History Metrics Supplementary material Preview PDF Abstract Driven by deep-learning approaches, computational protein design is advancing rapidly, and it is now possible to generate many de novo protein structures quickly and robustly. This sets new frontiers for the field, including designing proteins that bind small molecules tightly and specifically, and understanding the non-covalent interactions that underpin such designs to make binding predictable and tunable. Here we address these challenges with a rapid physics-based computational method to generate isosteric and chemically complementary binding pockets for small-molecule targets in de novo designed proteins. We test this experimentally by constructing and characterizing binding proteins for several synthetic and natural chromophores. By evaluating only single-digit numbers of designs, the pipeline delivers stable proteins with pre-organized binding sites confirmed by X-ray crystallography, which bind the targets selectively with micromolar affinities or better. To illustrate the scope and applications of this approach, we incorporate distinct and coupled chromophore-binding sites in a two-domain de novo protein enabling controlled energy transfer between the two sites, and we develop a small de novo binding protein that can be used in live mammalian cells to visualize sub-cellular structures. Competing Interest Statement The authors have declared no competing interest. Footnotes Sections on design protocol and active sampling updated for clarity; Figures 2, 3, 4, 6 revised; author affiliations updated; SI updated. Funder Information Declared Biotechnology and Biological Sciences Research Council , BB/X003027/1 , BB/T008741/1 Engineering and Physical Sciences Research Council , EP/T012455/1 Leverhulme Trust , RGP-2021-049 Royal Society , URF\R\201007 Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license . View the discussion thread. Back to top Previous Next Posted May 23, 2026. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Rapid Assessment of Size, Shape, and Chemical Complementarity of Ligands for Computational Protein Design Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Rapid Assessment of Size, Shape, and Chemical Complementarity of Ligands for Computational Protein Design Rokas Petrenas , Katarzyna Ozga , Joel J. Chubb , Andrey V. Romanyuk , Dominic Alibhai , Jennifer J. McManus , Graham J. Leggett , Nigel S. Scrutton , Thomas A. A. Oliver , Derek N. Woolfson bioRxiv 2025.06.30.662286; doi: https://doi.org/10.1101/2025.06.30.662286 Share This Article: Copy Citation Tools Rapid Assessment of Size, Shape, and Chemical Complementarity of Ligands for Computational Protein Design Rokas Petrenas , Katarzyna Ozga , Joel J. Chubb , Andrey V. Romanyuk , Dominic Alibhai , Jennifer J. McManus , Graham J. Leggett , Nigel S. Scrutton , Thomas A. A. Oliver , Derek N. Woolfson bioRxiv 2025.06.30.662286; doi: https://doi.org/10.1101/2025.06.30.662286 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biophysics Subject Areas All Articles Animal Behavior and Cognition (7636) Biochemistry (17704) Bioengineering (13898) Bioinformatics (41967) Biophysics (21460) Cancer Biology (18599) Cell Biology (25525) Clinical Trials (138) Developmental Biology (13384) Ecology (19909) Epidemiology (2067) Evolutionary Biology (24326) Genetics (15613) Genomics (22512) Immunology (17740) Microbiology (40423) Molecular Biology (17191) Neuroscience (88645) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4825) Physiology (7646) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)