BIBLIOS

Document type
Book chapters


Title
Thermal Shift and Stability Assays of Disease-Related Misfolded Proteins Using Differential Scanning Fluorimetry

Participants in the publication
Tânia G. Lucas (Author)
BioISI - Biosystems & Integrative Sciences Institute
Cláudio M. Gomes (Author)
Dep. Química e Bioquímica
BioISI
Bárbara J. Henriques (Author)
Dep. Química e Bioquímica
BioISI

Summary
Systematic identification of buffer formulations and small molecule chaperones that improve the expression, stability, and storage of proteins with therapeutic interest has gained enormous importance in biochemical research as well as in biotechnology and biomedical applications. In particular, the biochemical characterization of disease-related proteins and their genetic variants that result in misfolding requires systematic determination of protein stability, screening of optimal buffer conditions for biophysical and structural studies, and in some cases, the identification of small molecule chaperones with the potential to ameliorate folding defects. Among the several techniques available, differential scanning fluorimetry (DSF) is currently an extensively employed screening and analysis method for thermal shift and protein stability assays. Here we describe a step-by-step generic protocol for fast characterization of protein thermal stability and analysis of stabilization in thermal-shift assays by additives, ligands and chemical chaperones using β-oxidation mitochondrial dehydrogenases as model. These enzymes are associated to inborn errors of metabolism caused by mutant variants with folding and stability defects for which we previously established folding correction afforded by their cognate cofactors and substrates. With this example we thus illustrate the potential applications of the method in screening small molecule folding correctors among metabolites, ligands, cofactors or candidate drugs with therapeutic potential in protein folding diseases.

Editor(s)
Cláudio M. Gomes


Institution
Biosystems & Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa, Portugal.

Where published
Protein Misfolding Diseases - Methods and Protocols

Publication Identifiers
ISSN - 1064-3745
ISBN - 9781493988198,9781493988204

Address
New York, NY

Publisher
Humana Press

Collection
Methods in Molecular Biology



Document Identifiers
URL - http://dx.doi.org/10.1007/978-1-4939-8820-4_16

Rankings
SCIMAGO Q3 (2018) - 0.605 - Molecular Biology

Keywords
Thermal stability Protein stability Pharmacological chaperones Enzyme Melting temperature Differential scanning fluorimetry Cofactors Chemical chaperones