Protein-adaptive differential scanning fluorimetry using conformationally responsive dyes

Taiasean Wu; Joshua C Yu; Arundhati Suresh; Zachary J Gale-Day; Matthew G Alteen; Amanda S Woo; Zoe Millbern; Oleta T Johnson; Emma C Carroll; Carrie L Partch; Denis Fourches; Nelson R Vinueza; David J Vocadlo; Jason E Gestwicki

doi:10.1038/s41587-024-02158-7

Protein-adaptive differential scanning fluorimetry using conformationally responsive dyes

Nat Biotechnol. 2024 May 14. doi: 10.1038/s41587-024-02158-7. Online ahead of print.

Authors

Taiasean Wu^{1

2}, Joshua C Yu¹, Arundhati Suresh¹, Zachary J Gale-Day¹, Matthew G Alteen³, Amanda S Woo¹, Zoe Millbern⁴, Oleta T Johnson², Emma C Carroll², Carrie L Partch⁵, Denis Fourches⁴, Nelson R Vinueza⁴, David J Vocadlo^{3

6}, Jason E Gestwicki^{7

8}

Affiliations

¹ Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA.
² Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, USA.
³ Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
⁴ Department of Textile Engineering, North Carolina State University, Raleigh, NC, USA.
⁵ Department of Chemistry, University of California, Santa Cruz, CA, USA.
⁶ Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
⁷ Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA. jason.gestwicki@ucsf.edu.
⁸ Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, USA. jason.gestwicki@ucsf.edu.

PMID: 38744946
DOI: 10.1038/s41587-024-02158-7

Abstract

Differential scanning fluorimetry (DSF) is a technique that reports protein thermal stability via the selective recognition of unfolded states by fluorogenic dyes. However, DSF applications remain limited by protein incompatibilities with existing DSF dyes. Here we overcome this obstacle with the development of a protein-adaptive DSF platform (paDSF) that combines a dye library 'Aurora' with a streamlined procedure to identify protein-dye pairs on demand. paDSF was successfully applied to 94% (66 of 70) of proteins, tripling the previous compatibility and delivering assays for 66 functionally and biochemically diverse proteins, including 10 from severe acute respiratory syndrome coronavirus 2. We find that paDSF can be used to monitor biological processes that were previously inaccessible, demonstrated for the interdomain allostery of O-GlcNAc transferase. The chemical diversity and varied selectivities of Aurora dyes suggest that paDSF functionality may be readily extended. paDSF is a generalizable tool to interrogate protein stability, dynamics and ligand binding.