Alternate 13C−12C Labeling for Complete Mainchain Resonance Assignments using Cα Direct-Detection with Applicability Toward Fast Relaxing Protein Systems
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- 作者：Koh Takeuchi ; Zhen-Yu J. Sun ; Gerhard Wagner
- 刊名：Journal of the American Chemical Society
- 出版年：2008
- 出版时间：December 24, 2008
- 年：2008
- 卷：130
- 期：51
- 页码：17210-17211
- 全文大小：170K
- 年卷期：v.130,no.51(December 24, 2008)
- ISSN：1520-5126
文摘
Experiments that use direct 13C detection and take advantage of the slower relaxation of 13C magnetization compared to 1H offer an attractive strategy for extending the limits of NMR to include larger, highly dynamic, or paramagnetic proteins. Because carbonyl carbons (13C′) suffer from serious relaxation enhancement as a consequence of their large chemical shift anisotropy, deuterated α carbons are the preferred nuclei for 13C detection in large and/or fast relaxing systems. However, direct detection of 13Cα is not straightforward owing to the presence of one-bond 13C−13C couplings with 13C′ and 13Cβ that split the signals into multiples and hence reduce the sensitivity. Here we present the use of 13C enrichment at alternating carbon sites and deuteration at the Cα position to overcome these difficulties. The desired labeling pattern is achieved by expressing the protein in E.coli in D2O with either [2-13C] or [1,3-13C] glycerol as the carbon source. With this labeling strategy, we show that complete assignment of the main chain (including prolyl residues) can be achieved with a single CaN HSQC experiment. This approach offers advantages for the detection of NMR signals from sites with fast nuclear relaxation and offers promise for investigations of larger proteins and/or protein complexes that are inaccessible by proton-detected experiments.