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On-Resin Assembly of a Linkerless Lanthanide(III)-Based Luminescence Label and Its Application to the Total Synthesis of Site-Specifically Labeled Mechanosensitive Channels

Becker, Christian F. W. and Clayton, Daniel and Shapovalov, George and Lester, Henry A. and Kochendoerfer, Gerd G. (2004) On-Resin Assembly of a Linkerless Lanthanide(III)-Based Luminescence Label and Its Application to the Total Synthesis of Site-Specifically Labeled Mechanosensitive Channels. Bioconjugate Chemistry, 15 (5). pp. 1118-1124. ISSN 1043-1802. doi:10.1021/bc0498828. https://resolver.caltech.edu/CaltechAUTHORS:20170131-075938701

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Abstract

A synthesis strategy for the on-resin assembly of luminescent lanthanide chelates from commercially available compounds was developed. Advantages of the approach include the absence of spacers between the metal ion and the attachment site, and the compatibility with typical chemical protein synthesis protection schemes. Methoxycoumarin-labeled lysine and tris(tert-butyl)-DOTA were consecutively coupled with high efficiency to a free amino group in otherwise fully protected peptide segments using standard peptide synthesis methods. Addition of stoichiometric amounts of Tb3+ to the modified, cleaved, and purified peptides yielded the desired lanthanide chelate. Incorporation of this label into a chemically synthesized, full-length mechanosensitive channel of large conductance (MscL) of E. coli and subsequent reconstitution into vesicles resulted in a functional mechanosensitive channel of comparable conductance to the wild-type channel. However, this channel required increased suction to gate. Excitation of the antenna molecule methoxycoumarin at 336 nm resulted in an emission spectrum typical for Tb3+ and a luminescence lifetime of 0.67 ms. The location of the probe close to the backbone of this protein may provide precise information about conformational changes during channel opening from LRET studies.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bc0498828DOIArticle
http://pubs.acs.org/doi/abs/10.1021/bc0498828PublisherArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2004 American Chemical Society. Received 19 May 2004. Revised Manuscript Received July 15, 2004. Published online 24 August 2004. Published in print 1 September 2004. This work was supported by an NIH Program Project Grant, GM-062532.
Funders:
Funding AgencyGrant Number
NIHGM-062532
Issue or Number:5
DOI:10.1021/bc0498828
Record Number:CaltechAUTHORS:20170131-075938701
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170131-075938701
Official Citation:On-Resin Assembly of a Linkerless Lanthanide(III)-Based Luminescence Label and Its Application to the Total Synthesis of Site-Specifically Labeled Mechanosensitive Channels Christian F. W. Becker, Daniel Clayton, George Shapovalov, Henry A. Lester, and Gerd G. Kochendoerfer Bioconjugate Chemistry 2004 15 (5), 1118-1124 DOI: 10.1021/bc0498828
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73843
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:31 Jan 2017 17:53
Last Modified:11 Nov 2021 05:22

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