Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide–MHC complexes

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 measuremen...

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Bibliographic Details
Published in:Journal of immunological methods 2016-05, Vol.432, p.95-101
Main Authors: Hellman, Lance M., Yin, Liusong, Wang, Yuan, Blevins, Sydney J., Riley, Timothy P., Belden, Orrin S., Spear, Timothy T., Nishimura, Michael I., Stern, Lawrence J., Baker, Brian M.
Format: Article
Language:English
Subjects:
Binding affinity
Circular Dichroism
Differential scanning fluorimetry
Fluorometry - methods
HLA-A2 Antigen - chemistry
HLA-A2 Antigen - metabolism
HLA-DR1 Antigen - chemistry
HLA-DR1 Antigen - metabolism
Hot Temperature
Humans
Kinetic stability
Kinetics
MHC protein
Peptides - chemistry
Peptides - metabolism
Protein Binding
Protein Denaturation
Protein Stability
Thermal stability
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Summary: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. •The thermal stability of peptide/MHC complexes correlates with peptide affinity.•Differential scanning fluorimetry permits rapid assessment of peptide/MHC stability.•Substantially lower volume requirements of DSF provides for high throughput.•DSF also allows for measurements of peptide/MHC kinetic stability/dissociation rates.•Complexities not detectable with other binding assays can be observed with DSF.
ISSN:0022-1759
1872-7905
DOI:10.1016/j.jim.2016.02.016