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Selective Dye-Labeling of Newly Synthesized Proteins in Bacterial Cells

Beatty, Kimberly E. and Xie, Fang and Wang, Qian and Tirrell, David A. (2005) Selective Dye-Labeling of Newly Synthesized Proteins in Bacterial Cells. Journal of the American Chemical Society, 127 (41). pp. 14150-14151. ISSN 0002-7863. https://resolver.caltech.edu/CaltechAUTHORS:20150116-102630846

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Abstract

We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell.


Item Type:Article
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URLURL TypeDescription
http://dx.doi.org/10.1021/ja054643WDOIArticle
http://pubs.acs.org/doi/abs/10.1021/ja054643wPublisherArticle
http://pubs.acs.org/doi/suppl/10.1021/ja054643wPublisherSupplemental material
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
Additional Information:Copyright © 2005 American Chemical Society. Published In Issue October 19, 2005. Publication Date (Web): September 24, 2005. Received July 12, 2005. Acknowledgment. We thank Christopher W. Waters for help with microscopy, Mona Shahgholi for assistance with mass spectrometry, and Christina Smolke for use of her transilluminator. We are grateful to A. James Link for gifts of 4 and pQE30-Barstar, and to Marissa Mock and Sanne Schoffelen for advice and assistance. This work was supported by a Fannie and John Hertz Foundation Fellowship to K.E.B., by the National Institutes of Health, and by the Beckman Institute at Caltech. Supporting Information Available: Experimental protocols.
Funders:
Funding AgencyGrant Number
Fannie and John Hertz Foundation FellowshipUNSPECIFIED
NIHUNSPECIFIED
Beckman Institute at CaltechUNSPECIFIED
Issue or Number:41
Record Number:CaltechAUTHORS:20150116-102630846
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150116-102630846
Official Citation: Selective Dye-Labeling of Newly Synthesized Proteins in Bacterial Cells Kimberly E. Beatty,†, Fang Xie,‡, Qian Wang,‡ and, and David A. Tirrell*,† Journal of the American Chemical Society 2005 127 (41), 14150-14151
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53816
Collection:CaltechAUTHORS
Deposited By: Katherine Johnson
Deposited On:16 Jan 2015 18:47
Last Modified:03 Oct 2019 07:52

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