De novo helical peptides as target sequences for a specific, fluorogenic protein labelling strategy

Abstract

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.

A Green BODIPY‐Based, Super‐Fluorogenic, Protein‐Specific Labelling Agent

“…Maltose-binding protein (MBP) was chosen as ah ighly soluble test protein and the dC10a tag was fused to its Cterminus,asdescribed previously,togive MBP-dC10a. [22] The Cys residues of the dC10a tag are separated by % 10 , corresponding roughly to the distance between the electrophilic carbons of the maleimide groups of YC23.Inthis way, the dC10a tag presents aunique complementary dithiol motif for the bimolecular reaction with both maleimide groups of a meta-dimaleimide phenyl moiety.M BP-dC10a was expressed in bacteria and purified as described previously. [22] Prior to reaction with test protein, fluorogen YC23 showed negligible background fluorescence (Figure 1, black line on x-axis).…”

“…[22] The Cys residues of the dC10a tag are separated by % 10 , corresponding roughly to the distance between the electrophilic carbons of the maleimide groups of YC23.Inthis way, the dC10a tag presents aunique complementary dithiol motif for the bimolecular reaction with both maleimide groups of a meta-dimaleimide phenyl moiety.M BP-dC10a was expressed in bacteria and purified as described previously. [22] Prior to reaction with test protein, fluorogen YC23 showed negligible background fluorescence (Figure 1, black line on x-axis). This indicates that even with amethoxymaleimide group,w hose LUMO is of higher energy than an unsubstituted maleimide group,q uenching can still be very efficient.…”

“…[20,21] We have developed ac omplementary strategy for covalent, fluorogenic protein labelling that relies on the quenching properties of maleimides and their specific fluorogenic addition reaction (FlARe) with as mall, genetically encoded dicysteine peptide tag. [22][23][24][25][26][27] In the FlARe method, aPOI is genetically fused to ashort peptide tag (dC10a)that presents two Cys residues separated by two turns of an a-helix (ca. 10 ).…”

“…Cysteine is an underrepresented amino acid, and few proteins present two reactive Cys residues on their surface. [22] TheC ys residues of dC10a have been shown to be particularly reactive,p resumably because they exist more predominantly in their reactive thiolate forms. [22] Furthermore,b ecause the Cys residues are held apart by the secondary structural motif of the a-helix, they are less prone to formation of disulphide bonds;t his allowed the dC10a tag to be labelled [22] in the oxidizing environment of ac ell surface.T his spatial arrangement also favours their rapid reaction [22] with ac omplementary synthetic fluorogenic reagent, thereby turning on its fluorescence while labelling the POI.…”

Diversity‐Oriented Peptide Stapling: A Third Generation Copper‐Catalysed Azide–Alkyne Cycloaddition Stapling and Functionalisation Strategy

“…The groups of Timmerman, [12] Keillor [13] and Dawson [14] have reportede xamples of functionalised staples that rely on the alkylation of bis-cysteine-containing peptides. The ideal position for functionalisings tapled peptidesi so nthe staple, thereby positioninga dded functionality away from the biological target.…”