In‐depth interrogation of protein thermal unfolding data with MoltenProt

Abstract

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm, should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X‐ray crystallography and cryo‐electron microscopy, screening for small‐molecule binding partners and comparison of effects of point mutations.

Cyclohexyl-α maltoside as a highly efficient tool for membrane protein studies

“…An XLSX file containing the processed nanoDSF data was exported from PR.ThermControl and used for further analysis to extract the melting (T m ) and onset denaturation (T onset ) temperatures ( Kotov et al., 2019 ). Normalization of curves to calculate the fraction of unfolded protein was performed using the MoltenProt software ( Kotov et al., 2021 ).…”

eSPC: an online data-analysis platform for molecular biophysics

“…MoltenProt is a tool to estimate thermodynamic parameters from melting curves that was recently developed as a desktop application (Kotov et al, 2021). An example of a practical implementation of this module would be the screening of integral membrane-protein (IMP) stability in detergent or membrane-like environments (Kotov et al, 2019).…”

“…Users can upload their raw data and extract information from their DSF and MST experiments using three online tools. MoltenProt has been designed to evaluate the stability of a protein sample (Kotov et al, 2021) and FoldAffinity to determine equilibrium dissociation constants (K d ) from DSF experiments (Niebling et al, 2021). Both are now available in our EMBL Sample Preparation and Characterization (eSPC) online data-analysis platform.…”

An “Onion‐like” Model of Protein Unfolding: Collective versus Site Specific Approaches

“…The method uses aromatic residues, mainly Trp, but also Tyr, as intrinsic probes to monitor protein unfolding. [5,23] Thanks to their amphipathic character, these residues have a good occurrence in the hydrophobic core of proteins. [24] Protein unfolding and gradual exposure of buried Trp (or Tyr) to the solvent result in intensity change and/or red-shift of the fluorescence emission spectrum, which can then serve as a reporter of the global unfolding.…”

“…Intrinsic fluorescence spectroscopy and its high‐throughput ramifications, such as modified nano Differential Scanning Fluorimetry method (NanoDSF), can in principle be used to follow unfolding at a single residue level, but only under special circumstances. The method uses aromatic residues, mainly Trp, but also Tyr, as intrinsic probes to monitor protein unfolding [5,23] . Thanks to their amphipathic character, these residues have a good occurrence in the hydrophobic core of proteins [24] .…”