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Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells


A crucial step in the immune response is the binding of antigenic peptides to major histocompatibility complex (MHC) proteins. Class II MHC proteins present their bound peptides to CD4+ T cells, thereby helping to activate both the humoral and the cellular arms of the adaptive immune response. Peptide loading onto class II MHC proteins is regulated temporally, spatially and developmentally in antigen-presenting cells1. To help visualize these processes, we have developed a series of novel fluorogenic probes that incorporate the environment-sensitive amino acid analogs 6-N,N-dimethylamino-2-3-naphthalimidoalanine and 4-N,N-dimethylaminophthalimidoalanine. Upon binding to class II MHC proteins these fluorophores show large changes in emission spectra, quantum yield and fluorescence lifetime. Peptides incorporating these fluorophores bind specifically to class II MHC proteins on antigen-presenting cells and can be used to follow peptide binding in vivo. Using these probes we have tracked a developmentally regulated cell-surface peptide-binding activity in primary human monocyte-derived dendritic cells.

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Figure 1: 4-DAPA and 6-DMNA can be modeled into the P1 pocket of DR1 without major distortion.
Figure 2: Spectral properties of 6-DMNA and 4-DAPA peptides free in solution and bound to DR1.
Figure 3: Structural and functional characterization of (4-DAPA)-HA peptide.
Figure 4: (6-DMNA)-peptide binds specifically to DR molecules expressed on live cells.
Figure 5: (6-DMNA)-peptide binds to human monocyte-derived DCs in a maturation-dependent manner.

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We would like to thank M. Calvo-Calle for performing T-cell activation assays, R. Konz for assistance with flow cytometry experimentation and data interpretation, E. Lukovic and C. Painter for peptide synthesis, I. Strug for characterization of eluted peptides, E. Schreiter and Z. Zavala-Ruiz for helpful discussions, L. Lu for preparation of HLA-DR, J. Bill for the HA1.7 hybridoma, and the staff at the National Synchrotron Light Source (NSLS). Diffraction data for this study were measured at beamline X25 of the NSLS, support for which comes principally from the Office of Biological and Environmental Research and the Office of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources of the US National Institutes of Health. This work was supported by NIH-AI38996 (L.J.S.) and NSF CHE-0414243 (B.I.).

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Authors and Affiliations



P.V. performed the biochemical, cellular and spectroscopic studies, M.S. synthesized the fluorogenic amino acids and performed the chemical stability studies, T.N. determined the crystal structure, O.B. and S.C. performed biophysical and immunological assays, and P.V., M.S., B.I. and L.J.S wrote the manuscript.

Corresponding authors

Correspondence to Barbara Imperiali or Lawrence J Stern.

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Competing interests

There is a patent application pending (filed 13 April 2005) for the environment-sensitive amino acids originally introduced in J. Am. Chem. Soc. 127, 1300–1306 (2005).

Supplementary information

Supplementary Fig. 1

Absorbance and fluorescence spectra. (PDF 149 kb)

Supplementary Fig. 2

Peptide binding takes place primarily on the cell surface. (PDF 83 kb)

Supplementary Fig. 3

Open forms of (4-DAPA) and (4-DMNA) peptides. (PDF 267 kb)

Supplementary Table 1

Binding affinity of fluorogenic peptides. (PDF 27 kb)

Supplementary Table 2

(6-DMNA)-RSMA4L fluorescence intensity in different solvents. (PDF 44 kb)

Supplementary Table 3

Fluorescence lifetimes for free and DR-bound peptides. (PDF 74 kb)

Supplementary Table 4

Data collection and refinement statistics (molecular replacement). (PDF 71 kb)

Supplementary Methods

Preparation and analysis of ring-open forms; crystal structure; MHC-peptide binding assay; T-cell activation; fluorescence lifetime measurement. (PDF 60 kb)

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Venkatraman, P., Nguyen, T., Sainlos, M. et al. Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells. Nat Chem Biol 3, 222–228 (2007).

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