Biology subjects

Lampinen, V.

Publications and source records attributed to Lampinen, V..

2 recordsLinked to original sources

bioRxiv2026

Global analysis of thermal and chemical denaturation using CheMelt: Thermodynamic dissection of highly thermostable de novo designed proteins

De novo protein design often produces thermostable proteins that denature above 100 {degrees}C, which complicates the analysis of their stability. Thermostable proteins can be unfolded by combined chemical and thermal denaturation followed by global analysis of multiple melting curves. Here, we have developed CheMelt, a new online tool for global analysis of unfolding data via an intuitive graphical user interface. We use nanoscale differential scanning fluorimetry followed by CheMelt data analysis to dissect the combined thermal and chemical denaturation of thirty-five de novo designed protein binders. Thirteen present sufficient fluorescence changes to extract thermodynamic parameters of unfolding. These de novo designed proteins have systematically lower {Delta}Cp and m-values than comparable natural proteins. We show that a high thermostability of a designed protein does not necessarily imply a high equilibrium stability, and demonstrate the potential of CheMelt in dissecting thermodynamic properties for protein design and engineering. CheMelt can be accessed at https://spc.embl-hamburg.de/app/chemelt

biophysics↗

bioRxiv2025

Screening de novo designed protein binders in unpurified lysate using flow induced dispersion analysis

Computational protein design can create binders against targets of interest, but identifying binders with sufficient affinity still requires biochemical screening of many designs. In this work, we test flow-induced dispersion analysis (FIDA) as a method for screening binders in a time and cost-effective manner. FIDA uses Taylor dispersion analysis to determine the hydrodynamic radius of fluorescently labelled biomolecules and their complexes. Here, we use FIDA to assess the binding of RFdiffusion-designed protein binders against the small helical peptide ALFA-tag and the GK domain of PSD-95. Successful binders can be identified in a single measurement using heat-treated bacterial lysates allowing rapid identification of binders with high affinity, and thermostability. Subsequent titration experiments show micromolar affinities for ALFA-tag binders and nanomolar affinities for GK domain binders. The lack of immobilization, the minimal sample volume, and the compatibility with complex biological samples, positions FIDA as a valuable tool for the screening and characterization of computationally generated protein binders.

biophysics↗