Efficient Site-Specific Labeling of Proteins via Cysteines

Kim, Young-Gyu; Ho, Sam; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, F.; Weiss, Shimon

doi:10.1021/bc7002499

Cited by 247 publications

(199 citation statements)

References 32 publications

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“…As translocation substrate served a T61C variant of the HiPIP precursor, which was fluorescence-labeled by modification of the cysteine with fluorescein-5-maleimide using standard protocols for maleimidecoupling (61). HiPIP precursor was refolded from inclusion bodies and purified as described previously (36).…”

Section: Methodsmentioning

confidence: 99%

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

Hou¹,

Heidrich²,

Mehner-Breitfeld

et al. 2018

Journal of Biological Chemistry

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The twin-arginine translocation (Tat) system that comprises the TatA, TatB, and TatC components transports folded proteins across energized membranes of prokaryotes and plant plastids. It is not known, however, how the transport of this protein cargo is achieved. Favored models suggest that the TatA component supports transport by weakening the membrane upon full translocon assembly. Using Escherichia coli as model organism, we now demonstrate in vivo that the N-terminus of TatA can indeed destabilize the membrane, resulting in a lowered membrane energization in growing cells. We found that in full-length TatA, this effect is counterbalanced by its amphipathic helix. Consistent with these observations, the TatA N-terminus induced proton leakage in vitro, indicating membrane destabilization. Fluorescence quenching data revealed that substrate binding causes the TatA hinge region and the N-terminal part of the TatA amphipathic helix to move toward the membrane surface. In the presence of TatBC, substrate binding also reduced the exposure of a specific region in the amphipathic helix, indicating a participation of TatBC. Of note, the substrate-induced reorientation of the TatA amphipathic helix correlated with detectable membrane weakening. We therefore propose a two-state model in which membranedestabilizing effects of the short TatA membrane anchor are compensated by the membraneimmersed N-terminal part of the amphipathic helix in a resting state. We conclude that substrate binding to TatABC complexes switches the position of the amphipathic helix, which locally weakens the membrane on demand to allow substrate translocation across the membrane.The Tat system serves to transport folded proteins in bacteria, archaea, plant plastids, and possibly plant mitochondria (1-4). In Escherichia coli, the Tat system consists of TatA, TatB, and TatC components. TatA and TatB are similar, and two-component "minimal" Tat systems exist in which TatA exerts functions of TatA and TatB (5). TatA/B components are N-terminally membraneanchored by a very short hydrophobic transmembrane domain (TMD), which is followed by a short hinge region, an amphipathic helix (APH), and a variable C-terminal domain (6). TatC is a polytopic membrane protein with six TMDs (7,8). TatB tightly interacts with TatC (9, 10). TatA associates with these TatBC complexes and is thought to permeabilize the membrane for protein transport (11)(12)(13)(14). TatBC complexes recognize and tightly bind the signal peptides of the cargo proteins throughout the translocation process (15,16).The mechanism by which this translocation is achieved must be unusual and is not understood (17). A currently favored model suggests that the N-termini of multiple TatA molecules at the translocon site weaken the membrane upon substrate-binding, thereby permitting a TatCmediated pulling of the substrate through the Control of membrane-weakening by TatA 2 destabilized membrane ("membrane-weakening and pulling mechanism", 18). Molecular dynamics simulations support this view ...

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Section: Methodsmentioning

confidence: 99%

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

Hou¹,

Heidrich²,

Mehner-Breitfeld

et al. 2018

Journal of Biological Chemistry

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“…[5][6][7] For instance, fluorophore-conjugated maleimides have been employed for the modification of cysteine residues. 8,9) Recently, 2-bromomaleimide and 2,3-dibromomaleimide were developed as novel cysteine-labeling reagents, which react with thiol in an addition-elimination sequence (nucleophilic substitution) to afford thiomaleimide. [10][11][12][13][14][15][16][17][18] In an organic solvent such as tetrahydrofuran (THF), 2,3-dibromomaleimide also reacts with amine, affording 2-amino-3-bromomaleimide as an amine-conjugation product.…”

Section: Molecular Design Model Reaction and Spectrometric Analysismentioning

confidence: 99%

Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group for Use in Affinity Labeling

Chiba

Hashimoto

Yamaguchi

2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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Chemical probes comprising a ligand moiety, a reactive group (e.g. epoxide, haloacetyl or photoreactive group) and a tag unit (e.g. fluorophore or radioisotope) are widely used in affinity labeling to identify the target proteins of bioactive molecules. However, design and synthesis of highly functionalized chemical probes are often time-consuming. In this paper, we propose a simple design strategy for chemical probes bearing a small 2,3-dichloromaleimide (diCMI) unit, which serves as a combined reactive group and tag unit by reacting with a nucleophilic lysine residue near the ligand-binding site of the target protein to generate the 2-amino-3-chloromaleimide fluorophore. Model ligand-protein experiments confirmed that the diCMI unit has suitable reactivity and fluorogenic capability for efficient affinity labeling.Key words affinity labeling; dichloromaleimide; protein modification Affinity labeling is a powerful method to label and visualize target proteins of bioactive molecules.1,2) This method generally utilizes a chemical probe composed of a ligand moiety, a reactive group and a tag unit (Fig. 1a). The reactive group serves to form a covalent bond with the target protein. For efficient and specific target labeling, the reactive group should be stable in aqueous media, have low reactivity towards non-target molecules, and react appropriately with the target protein after ligand-target binding. The tag unit serves to visualize the target protein. Fluorophores are often used as a tag unit because of the ease of detection. However, a major obstacle to affinity labeling is often synthesis of the highly functionalized chemical probe. More importantly, the ligand must retain its binding affinity after introduction of these two functional groups (reactive group and tag unit). For these reasons, a small alkyne tag is often used in affinity labeling, since it can be subsequently conjugated with a detection unit (e.g. azide-fluorophore, azide-biotin), despite the inconvenience of the additional conjugation step(s).3) We recently reported a small alkoxy nitrobenzoxadiazole (O-NBD, 180 Da) unit as a fluorogenic reactive group for affinity labeling.4) Here, we show that 2,3-dichloromaleimide (diCMI, 164 Da) can serve as an even smaller fluorogenic reactive group (Fig. 1b). Results and DiscussionMolecular Design, Model Reaction and Spectrometric Analysis The maleimide motif is a highly reactive Michael acceptor and has been widely used for chemical modification of thiols of biomolecules.5-7) For instance, fluorophore-conjugated maleimides have been employed for the modification of cysteine residues. 8,9) Recently, 2-bromomaleimide and 2,3-dibromomaleimide were developed as novel cysteine-labeling reagents, which react with thiol in an addition-elimination sequence (nucleophilic substitution) to afford thiomaleimide. 10-18)In an organic solvent such as tetrahydrofuran (THF), 2,3-dibromomaleimide also reacts with amine, affording 2-amino-3-bromomaleimide as an amine-conjugation product. Interestingly, 2-aminomaleimides we...

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“…9 Fluorescent probes incorporated into peptides should combine strong changes of their emission upon membrane binding with relatively small size in order to minimally perturb the peptide structure and report on the environment as close as possible to the peptide backbone. The classical bioconjugation strategies for fluorescent labeling of peptides are based on coupling of sulfhydryl-and amino-reactive dyes with cysteine 10 and lysine 11 amino acids, respectively. They were successfully applied to graft environment-sensitive dyes, such as NBD, 12 Dansyl, 13,14 and Prodan, 15 to peptides for site-specific probing of the peptide−membrane interactions.…”

Section: ■ Introductionmentioning

confidence: 99%

Dual-Fluorescence l-Amino Acid Reports Insertion and Orientation of Melittin Peptide in Cell Membranes

et al. 2013

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Monitoring insertion and orientation of peptides in situ on cell membranes remains a challenge. To this end, we synthesized an L-amino acid (AFaa) containing a dualfluorescence dye of the 3-hydroxyflavone family, as a side chain. In contrast to other labeling approaches using a flexible linker, the AFaa fluorophore, introduced by solid phase synthesis into desired position of a peptide, is attached closely to its backbone with well-defined orientation, and, therefore, could reflect its localization in the membrane. This concept was validated by replacing the leucine-9 (L9) and tryptophan-19 (W19) residues by AFaa in melittin, a well-studied membrane-active peptide. Due to high sensitivity of AFaa dual emission to the environment polarity, we detected a much deeper insertion of L9 peptide position into the bilayer, compared to the W19 position. Moreover, using fluorescence microscopy with a polarized light excitation, we found different orientation of AFaa at L9 and W19 positions of melittin in the bilayers of giant vesicles and cellular membranes. These results suggested that in the natural membranes, similarly to the model lipid bilayers, melittin is preferentially oriented parallel to the membrane surface. The developed amino acid and the proposed methodology will be of interest to study other membrane peptides.

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Efficient Site-Specific Labeling of Proteins via Cysteines

Cited by 247 publications

References 32 publications

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group for Use in Affinity Labeling

Dual-Fluorescence l-Amino Acid Reports Insertion and Orientation of Melittin Peptide in Cell Membranes

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