NDLI: An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

Content Provider	PubMed Central
Author	Velyvis, Algirdas Ruschak, Amy M. Kay, Lewis E.
Editor	Ramón, Campos-olivas
Copyright Year	2012
Abstract	NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby 13CH3 methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-2H; β−2H;γ-13C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr 13C,1H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[13C,1H]-Thr.
Related Links	http://dx.doi.org/10.1371/journal.pone.0043725
Starting Page	43725
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	9
Volume Number	7
Language	English
Publisher	Public Library of Science
Publisher Date	2012-09-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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