De novo helical peptides as target sequences for a specific, fluorogenic protein labelling strategy.
New methods are needed to selectively label proteins in a manner that minimally perturbs their structures and functions. We have developed a 'small molecule'-based labelling technique that relies on the use of dimaleimide fluorogens that react with a target peptide sequence that presents appropriately spaced, solvent-exposed Cys residues. The thiol addition reaction between target sequence and dimaleimide fluorogen restores the latent fluorescence of the latter and results in the covalent fluorescent labelling of the protein of interest (J. Guy, K. Caron, S. Dufresne, S. W. Michnick, W. G. Skene and J. W. Keillor, J. Am. Chem. Soc., 2007, 129, 11969-11977). We demonstrated the proof-of-principle of this method previously, using a dicysteine mutant of the helical protein Fos (S. Girouard, M.-H. Houle, A. Grandbois, J. W. Keillor and S. W. Michnick, J. Am. Chem. Soc., 2005, 127, 559-566). Herein, we present the design of a novel peptide sequence presenting two Cys residues separated by two turns of an alpha-helix. The secondary structure of this sequence was confirmed by CD spectroscopy, before and after the fluorescent labelling reaction. A new series of di(3-methylmaleimide) fluorogens was prepared and kinetically evaluated, tuning their reactivity toward the target sequence. Attempts were made to increase the reactivity of the parent target sequence by rational design; however, the introduction of basic His residues in the vicinity of one or more Cys residues did not have the desired effect. Finally, epidermal growth factor receptors bearing the de novo target sequence were specifically labelled with a di(3-methylmaleimide) fluorescein fluorogen, validating our method for specific cell-surface labelling of proteins. A wide variety of fluorogen and peptide designs can be envisioned with potential applications to multiplexed labelling for the study of temporal and spatial dynamics of protein expression.
Guy
Julia
J
Département de chimie, Université de Montréal, Montréal, QCH3C3J7, Canada.
Castonguay
Roselyne
R
Campos-Reales Pineda
Natalhie B
NB
Jacquier
Valérie
V
Caron
Karine
K
Michnick
Stephen W
SW
Keillor
Jeffrey W
JW
D000595:Amino Acid Sequence; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008301:Maleimides / Q000737:chemistry; D058619:Maltose-Binding Proteins; D018613:Microscopy, Confocal; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D008301:Maleimides; D058619:Maltose-Binding Proteins; D010455:Peptides; D033902:Periplasmic Binding Proteins; D011506:Proteins; D016760:Proto-Oncogene Proteins c-fos; C043592:maleimide; D066246:ErbB Receptors; D003545:Cysteine