Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes.
When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to the cell surface. The peptides are cleaved to the precise lengths required for MHC-I binding and detection by T cells. We have developed fluorescent indicators to study the cleavage of peptides in living cells as they are transported from the endoplasmic reticulum to the Golgi apparatus. Specific viral peptides known to be "trimmed" prior to cell surface presentation were labeled with two dyes undergoing fluorescence resonance energy transfer (FRET). When these fluorescent peptides were proteolytically processed in living cells, FRET was halted, so that each labeled fragment and the intact peptide exhibited different fluorescence spectra. Such fluorescent cleavage indicators can be used to study a wide range of biological behaviors dependent on peptide or protein cleavage. However, labeling a peptide with two dyes at precise positions can present a major obstacle to using this technique. Here, we describe two approaches for preparing doubly labeled cleavage indicator peptides. These methods are accessible to researchers using standard laboratory techniques or, for more demanding applications, through cooperation with commercial or core peptide synthesis services using minor modifications of standard synthetic procedures.
Bark
S J
SJ
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Hahn
K M
KM
D017951:Antigen Presentation; D001692:Biological Transport; D010450:Endopeptidases / Q000302:isolation & purification*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry
D005456:Fluorescent Dyes; D010455:Peptides; D012235:Rhodamines; D013388:Succinimides; C073461:tetramethylrhodamine hydroxysuccinimide; D010450:Endopeptidases