Title
Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide–MHC complexes
Author
Lance M. Hellman, Liusong Yin, Yuan Wang, Sydney J. Blevins, Timothy P. Riley, Orrin S. Belden, Timothy T. Spear, Michael I. Nishimura, Lawrence J. Stern, Brian M. Baker
Year
2016
Journal
Journal of Immunological Methods
Abstract
Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics.
Instrument
J-815
Keywords
Circular dichroism, Protein folding, Protein denaturation, Secondary structure, Thermal stability, Thermodynamics, Pharmaceutical, Biochemistry