Advanced

Site-selective 13C labeling of proteins using erythrose

Weininger, Ulrich LU (2017) In Journal of Biomolecular NMR
Abstract

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13C and/or 1H, which is achieved in the most general way by using site-selectively 13C-enriched glucose (1- and 2-13C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we... (More)

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13C and/or 1H, which is achieved in the most general way by using site-selectively 13C-enriched glucose (1- and 2-13C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively 13C-enriched erythrose (1-, 2-, 3- and 4-13C) as a suitable precursor for 13C labeled aromatic side chains. We quantify 13C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the 13C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated 13C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective 13C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

(Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
epub
subject
keywords
Aromatic side chain, Isotope labeling, Protein dynamics, Relaxation
in
Journal of Biomolecular NMR
pages
10 pages
publisher
Springer
external identifiers
  • scopus:85014031823
  • wos:000399299600003
ISSN
0925-2738
DOI
10.1007/s10858-017-0096-7
language
English
LU publication?
yes
id
988a7399-8984-4713-a99f-f6ebfb9750e3
date added to LUP
2017-03-13 10:02:55
date last changed
2018-01-07 11:55:02
@article{988a7399-8984-4713-a99f-f6ebfb9750e3,
  abstract     = {<p>NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with <sup>13</sup>C and/or <sup>1</sup>H, which is achieved in the most general way by using site-selectively <sup>13</sup>C-enriched glucose (1- and 2-<sup>13</sup>C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively <sup>13</sup>C-enriched erythrose (1-, 2-, 3- and 4-<sup>13</sup>C) as a suitable precursor for <sup>13</sup>C labeled aromatic side chains. We quantify <sup>13</sup>C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the <sup>13</sup>C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated <sup>13</sup>C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective <sup>13</sup>C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.</p>},
  author       = {Weininger, Ulrich},
  issn         = {0925-2738},
  keyword      = {Aromatic side chain,Isotope labeling,Protein dynamics,Relaxation},
  language     = {eng},
  month        = {02},
  pages        = {10},
  publisher    = {Springer},
  series       = {Journal of Biomolecular NMR},
  title        = {Site-selective <sup>13</sup>C labeling of proteins using erythrose},
  url          = {http://dx.doi.org/10.1007/s10858-017-0096-7},
  year         = {2017},
}