In-depth interrogation of protein thermal unfolding data with MoltenProt.

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.

Structural and antigenic investigation of Usutu virus envelope protein domain III.

The mosquito-borne flavivirus Usutu virus (USUV) has recently emerged in birds and humans in Europe. Symptoms of a USUV infection resemble those of West Nile virus (WNV); further, the close antigenic relationship of domain III (DIII) of the USUV and WNV envelope (E) proteins has prevented the development of a reliable serological test to distinguish USUV from WNV. To begin to address this deficiency, we identified ten different sequence groups of DIII from 253 complete and 80 partial USUV genome sequences. We solved the DIII structures of four groups, including that of the outlying CAR-1969 strain, which shows an atypical DIII structure. Structural comparisons of the USUV DIII groups and the DIII of WNV bound to the neutralizing antibody E16 revealed why the E16 failed to neutralize all USUV strains tested except for USUV CAR-1969. The analyses allowed predictions to be made to engineer an antibody specific for USUV CAR-1969.