title: Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide-MHC complexes.
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.
authors.affiliation: Department of Chemistry & Biochemistry and the Harper Cancer Research Institute, A224 Harper Hall, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Lance M
authors.initials: LM
authors.lastname: Hellman
authors.affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Liusong
authors.initials: L
authors.lastname: Yin
authors.affiliation: Department of Chemistry & Biochemistry and the Harper Cancer Research Institute, A224 Harper Hall, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry & Biochemistry and the Harper Cancer Research Institute, A224 Harper Hall, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Sydney J
authors.initials: SJ
authors.lastname: Blevins
authors.affiliation: Department of Chemistry & Biochemistry and the Harper Cancer Research Institute, A224 Harper Hall, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Riley
authors.affiliation: Department of Chemistry & Biochemistry and the Harper Cancer Research Institute, A224 Harper Hall, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Orrin S
authors.initials: OS
authors.lastname: Belden
authors.affiliation: Department of Surgery and the Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Spear
authors.affiliation: Department of Surgery and the Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Michael I
authors.initials: MI
authors.lastname: Nishimura
authors.affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Stern
authors.affiliation: Department of Chemistry & Biochemistry and the Harper Cancer Research Institute, A224 Harper Hall, University of Notre Dame, Notre Dame, IN 46556, USA. Electronic address: brian-baker@nd.edu.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Baker
mesh_terms: D002942:Circular Dichroism; D005470:Fluorometry / Q000379:methods*; D015789:HLA-A2 Antigen / Q000737:chemistry / Q000378:metabolism*; D015798:HLA-DR1 Antigen / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability
chemical_list: C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D015798:HLA-DR1 Antigen; D010455:Peptides
keywords: Binding affinity; Differential scanning fluorimetry; Kinetic stability; MHC protein; Thermal stability