This communication describes a novel protein labeling method that uses a single amino-acid tag -N-terminal cysteine residue -and small-molecule probes carrying the cyanobenzothiazole unit for specific labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity in physiological conditions, and should offer an important alternative to currently available protein labeling methods. Graphical abstract KeywordsProtein Labeling; Condensation; Terminal Cysteine; Chemical ligation; Live-cell Imaging Site-specific labeling of proteins with molecular tags enables direct visualization of protein dynamics, localization and interactions in single living cells and is a powerful tool for studying structure and function of proteins. [1] Proteins of interest can be labeled by genetic fusions to fluorescent proteins, or chemical reactions with fluorescent dyes. Chemical labeling often employs a receptor protein, for example, a mutant of human O 6 -alkylguanine-* Fax: (+1) 650-736-7925, ; Email: jrao@stanford.edu, Homepage: http://raolab.stanford.edu Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript DNA transferase, [2a] and E. coli dihydrofolate reductase, [2b] that binds to or reacts with its fluorescently tagged ligand. [2] Alternatively, smaller tags such as short peptides can be labeled by selective binding to fluorogenic dyes [3] or by enzyme-catalyzed ligation to fluorescent probes. [4] Water-compatible chemical reactions can also be applied to protein labeling, such as the Staudinger reaction between the azides and triphenylphosphines, [5] the Huisgen cycloaddition or "Click chemistry" between the azides and alkynes, [6] the reaction between aldehydes (or ketones) and aminooxy containing reagents (or hydrazides). [7] Herein, we describe a water-compatible condensation reaction for labeling terminal cysteine residues on proteins in vitro and at the cell surface.N-terminal cysteine has been frequently used in protein engineering for site-specific labeling and modification. [8] Thioesters are commonly used in a ligation reaction with terminal cysteines, which proceeds through thioester and S-to N-acyl exchanges. [9] This native chemical ligation reaction has been successfully applied to protein semi-synthesis and labeling. [10] Our method to label the terminal cysteine on a protein is based on the condensation of 2-cyanobenzothiazole (CBT) and D-cysteine, a reaction used at the last step of the synthesis of D-luciferin a common substrate for firefly luciferase (reaction 1 in Scheme 1). [11] This reaction can proceed smoothly in aqueous solutions. We hypothesized that CBT could react with the terminal cysteine on a protein. If a fluorophore is conjugated to the CBT motif, this reaction should ligate a fluorescent label specifically to the terminal cysteine of the protein (reaction...
Rapid and sensitive assay of proteases and their inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. We developed a multiplexed assay system of proteases and their inhibition by measuring the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) on a glass slide. In this system, while the photoluminescence (PL) of donor QDs immobilized on a surface was quenched due to the presence of AuNPs (energy acceptor) in close proximity, the protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the protease assay. In comparison to the QD-dye system, the conjugate of the QD-AuNP gave rise to higher energy transfer efficiency, resulting in quantitative assay of proteases with more sensitivity. When matrix metalloproteinase, caspase, and thrombin were tested, a multiplexed assay was successfully achieved since the AuNP could be used as a common energy acceptor in conjunction with QDs having different colors. Our system is anticipated to find applications in the diagnosis of protease-related diseases and screening of potential drugs with high sensitivity in a high-throughput way.
This review demonstrates the detection of protease activity based on the energy transfer of quantum dots (QDs). By incorporation of varying protease substrates into designed QD probes both in fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) system, proteolytic activity led to changes in the energy transfer efficiency. Especially due to the superior properties of QDs, it can be served as an excellent probe for a multiplexed and high-throughput protease assay with high sensitivity. It is anticipated that the QD-based FRET/BRET probes will have a great potential for dissecting the fundamental roles of proteases and designing potential protease inhibitors as therapeutic drugs in biology and nanomedicine.
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