DOTA-Amide Lanthanide Tag for Reliable Generation of Pseudocontact Shifts in Protein NMR Spectra

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• 作者：Bim Graham ; Choy Theng Loh ; James David Swarbrick ; Phuc Ung ; James Shin ; Hiromasa Yagi ; Xinying Jia ; Sandeep Chhabra ; Nicholas Barlow ; Guido Pintacuda ; Thomas Huber ; Gottfried Otting
• 刊名：Bioconjugate Chemistry
• 出版年：2011
• 出版时间：October 19, 2011
• 年：2011
• 卷：22
• 期：10
• 页码：2118-2125
• 全文大小：861K
• 年卷期：v.22,no.10(October 19, 2011)
• ISSN：1520-4812

文摘

Structural studies of proteins and protein鈥搇igand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin.