Graduate School of Biomedical Sciences, Program in Biochemistry and Molecular Pharmacology; Department of Pathology
Amino Acids, Peptides, and Proteins | Biochemistry, Biophysics, and Structural Biology | Biological Factors | Hemic and Immune Systems | Immunology and Infectious Disease
Major Histocompatibility Complex class II (MHC-II) molecules bind peptides and present them to receptors on CD4+ T cells as part of the immune system's surveillance of pathogens and malignancy. In the absence of peptide, MHC-II equilibrates between peptide-receptive and peptide-averse conformations. The conversion between these forms has been postulated to be important in regulating cellular antigen presentation but has been difficult to study. In order to generate the MHC-II molecule HLA-DR1 in the peptide-receptive form, we designed and tested a series of photocleavable peptides that included the UV-sensitive 3-amino-3-(2-nitrophenyl)-propionate amino acid analog. They were intended to bind tightly to the HLA-DR1 MHC molecule, but to generate low-affinity fragments after UV exposure that would be released to yield HLA-DR1 in the peptide-receptive conformation. We were able to identify photocleavable peptides that bound tightly to HLA-DR1 and generated the peptide-receptive conformation after UV exposure. However, slow release of photocleaved peptide fragments from the binding site limited the rate of binding of an incoming labeled peptide and complicated kinetic measurements of the individual steps of the overall peptide binding reaction. Modification of the N-terminal region of the photocleavable peptide to reduce MHC-II pocket or H-bonding interactions allowed for generation of the peptide receptive form immediately after UV exposure with peptide fragments neither retained within the site nor interfering with binding of an incoming peptide. However this was achieved only at the expense of a substantial reduction in overall peptide binding affinity, and these peptides had such weak interaction with HLA-DR1 that they were easily exchanged by incoming peptide without UV exposure. These results show that photocleavable peptides can be used to generate peptide-receptive HLA-DR1 and to facilitate peptide exchange in generation of specific peptide-MHC-II complexes, but that usage of these peptides for kinetic studies can be constrained by slow fragment release.
Hydrogen bonding, Major histocompatibility complex, Fluorescence polarization, Binding analysis, T cell receptors, Tyrosine, Fluorescence competition, T cells
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Copyright: © 2018 Negroni, Stern. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
DOI of Published Version
PLoS One. 2018 Jul 2;13(7):e0199704. doi: 10.1371/journal.pone.0199704. eCollection 2018. Link to article on publisher's site
Negroni, Maria P. and Stern, Lawrence J., "The N-terminal region of photocleavable peptides that bind HLA-DR1 determines the kinetics of fragment release" (2018). Open Access Articles. 3532.
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