Engineering encodable lanthanide-binding tags into loop regions of proteins.
Engineering encodable lanthanide-binding tags into loop regions of proteins.
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DOI：
10.1021/ja104983t
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发表时间：
2011-02-02
影响因子：
15
通讯作者：
Schwalbe, Harald
中科院分区：
文献类型：
--
作者：
Barthelmes, Katja;Reynolds, Anne M.;Peisach, Ezra;Jonker, Hendrik R. A.;DeNunzio, Nicholas J.;Allen, Karen N.;Imperiali, Barbara;Schwalbe, Harald
稀土元素结合标记(LBT)是利用核磁共振光谱、X射线结晶学和发光研究来研究蛋白质结构、功能和动力学的有价值的工具。我们将LBT插入到模型蛋白IL-1β的三个不同的环位(标记为L、R和S)，并改变了LBT与蛋白质之间的间隔区长度(标记为1-3)。发光研究表明，所有九个结构都在低纳摩尔范围内与Tb3+紧密结合。插入LBT后融合蛋白没有显著变化，IL 1β-S1和IL 1β-L3结构的两个X射线晶体结构表明，其余结构的1H-15N-HSQC核磁共振谱与野生型IL 1β相比较。此外，在体外，LBT-loop IL1β蛋白与其天然结合伙伴的结合没有改变。仅用结合的稀土元素的信号，就成功地进行了X射线晶相分析。所有LBT-环结构的核磁共振波谱都可以测定到大的残留偶极耦合(RDC)，表明LBT-2系列刚性地结合到IL-1的β结构中。指定了环-lbt突变体β-R2的顺磁核磁共振谱，并根据RDC和伪接触位移(PCSS)计算了Δχ张量分量。利用顺磁约束计算了IL1β-R2结构的结构模型。目前的数据支持，当将可编码的LBT插入到已知结构或通过同源建模预测的蛋白质的环区时，可编码的LBT作为多功能生物物理标签。
Lanthanide-binding-tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β and varied the length of the spacer between the LBT and the protein (denoted 1-3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing 1H-15N-HSQC NMR spectra with wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop-constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated based on RDCs and pseudocontact shifts (PCSs). A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modelling.
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影响因子：
2.9
作者：
CLORE, GM;WINGFIELD, PT;GRONENBORN, AM
通讯作者：
GRONENBORN, AM
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.7
作者：
Feeney, J;Birdsall, B;Bayley, PM
通讯作者：
Bayley, PM
可变螺旋伸长作为调节RNA比对和运动耦合的工具。
DOI：
10.1016/j.jmr.2009.09.022
发表时间：
2010-01
期刊：
Journal of magnetic resonance (San Diego, Calif. : 1997)
影响因子：
--
作者：
Dethoff EA;Hansen AL;Zhang Q;Al-Hashimi HM
通讯作者：
Al-Hashimi HM
DOI：
10.1093/protein/12.3.189
发表时间：
1999-03-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Arico-Muendel, CC;Patera, A;Wolfson, AJ
通讯作者：
Wolfson, AJ