DOTA-M8: An Extremely Rigid, High-Affinity Lanthanide Chelating Tag for PCS NMR Spectroscopy
DOTA-M8: An Extremely Rigid, High-Affinity Lanthanide Chelating Tag for PCS NMR Spectroscopy
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DOI：
10.1021/ja903233w
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发表时间：
2009-10-21
影响因子：
15
通讯作者：
Grzesiek, Stephan
中科院分区：
文献类型：
--
作者：
Haussinger, Daniel;Huang, Jie-rong;Grzesiek, Stephan
提出了一种新的用于生物分子顺磁性标记的镧系螯合标记物（M8），其基于八重立体选择性甲基取代的DOTA，其可以通过单个二硫键共价连接到主体分子。DOTA支架的空间过度拥挤导致极其刚性、动力学和化学惰性的镧系元素螯合剂。其空间体积限制了标签相对于主体分子的运动。这些性质导致与泛素连接的Dy-M8的非常大的伪接触位移（>5 ppm）和残余偶极偶联（>20 Hz），这对于小的单点连接标签是前所未有的。这种大的伪接触位移应该是很好地检测，甚至对于较大的蛋白质和超过类似于50埃的距离。由于其极高的稳定性和镧系元素亲和力，M8可以在极端的化学或物理条件下使用，例如用于蛋白质变性的条件，或者当缓冲液或蛋白质不希望与过量的镧系元素离子反应时。
A new lanthanide chelating tag (M8) for paramagnetic labeling of biomolecules is presented, which is based on an eight-fold, stereoselectively methyl-substituted DOTA that can be covalently linked to the host molecule by a single disulfide bond. The steric overcrowding of the DOTA scaffold leads to an extremely rigid, kinetically and chemically inert lanthanide chelator Its steric bulk restricts the motion of the tag relative to the host molecule. These properties result in very large pseudocontact shifts (>5 ppm) and residual dipolar couplings (>20 Hz) for Dy-M8 linked to ubiquitin, which are unprecedented for a small, single-point-attachment tag. Such large pseudocontact shifts should be well detectable even for larger proteins and distances beyond similar to 50 angstrom. Due to its exceptionally high stability and lanthanide affinity M8 can be used under extreme chemical or physical conditions, such as those applied for protein denaturation, or when it is undesirable that buffer or protein react with excess lanthanide ions.