Wissett, O. | Search bioRxiv

bioRxiv2025

SurfDiff: protein surface profiling for selective or broadly reactive epitope prioritisation in binder and immunogen design

Rational selection of protein-surface patches - epitopes or ligandable sites - is essential for developing targeted antibodies, binding proteins, peptides, or small-molecule ligands, and for informing immunogen design in vaccines. We present SurfDiff, a structure- and sequence-informed framework for protein surface comparison. SurfDiff supports one-to-one and one-to-many comparisons by combining local structural alignments with physicochemically and spatially aware neighbourhood analysis. It assigns residue-level uniqueness and similarity scores, which can be aggregated into surface-level similarity, selectivity, and a discriminability score that captures conservation across desired targets while penalising similarity to undesired off-targets. These scores, calculated solely from the targets without any knowledge of the binders, reliably predict known experimental selectivity for diverse binders including small molecules, peptides, antibodies and antibody mimetics across viral antigens, cytokine isoforms, GPCR subtypes and serum albumin. They also correlate strongly with binding and neutralisation data across pathogen variants, and substantially outperform commonly used bioinformatic metrics such as sequence substitution matrices and structural similarity measures. SurfDiff provides a generalisable and interpretable approach to protein surface profiling, enabling selective or cross-reactive binder design, cross-species prioritisation, and facilitating immunogen selection. We make SurfDiff available as downloadable open-source software: (https://gitlab.developers.cam.ac.uk/ch/sormanni/surfdiff) and as a webserver: www-cohsoftware.ch.cam.ac.uk/index.php/surfdiff.

bioinformatics↗

bioRxiv2025

The SpyBLI cell-free pipeline for the rapid quantification of binding kinetics from crude samples

Accurate measurements of binding kinetics, encompassing equilibrium dissociation constant (KD), association rate (kon), and dissociation rate (ko,), are critical for the development and optimisation of high-affinity binding proteins. However, such measurements require highly purified material and precise ligand immobilisation, limiting the number of binders that can be characterised within a reasonable timescale and budget. Here, we present the SpyBLI method, a rapid and cost-effective biolayer interferometry (BLI) pipeline that leverages the SpyCatcher003-SpyTag003 covalent association, eliminating the need for both binder purification and concentration determination. This approach allows for accurate binding-kinetic measurements to be performed directly from crude mammalian-cell supernatants or cell-free expression mixtures. We also introduce a linear gene fragment design that enables reliable expression in cell-free systems without any PCR or cloning steps, allowing binding kinetics data to be collected in under 24 hours from receiving inexpensive DNA fragments, with minimal hands-on time. We demonstrate the methods broad applicability using a range of nanobodies and single-chain antibody variable fragments (scFvs), with affinity values spanning six orders of magnitude. By minimising sample preparation and employing highly controlled, ordered sensor immobilisation, our workflow delivers reliable kinetic measurements from crude mixtures without sacrificing precision. We expect that the opportunity to carry out rapid and accurate binding measurements in good throughput should prove especially valuable for binder engineering, the screening of next-generation sequencing-derived libraries, and computational protein design, where large numbers of potential binders for the same target must be rapidly and accurately characterised to enable iterative refinement and candidate selection.

biophysics↗