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New Lanthanide Chelating Tags for PCS NMR Spectroscopy with Reduction Stable, Rigid Linkers for Fast and Irreversible Conjugation to Proteins
Bioconjugate Chemistry ( IF 4.7 ) Pub Date : 2018-08-30 00:00:00 , DOI: 10.1021/acs.bioconjchem.8b00512
Thomas Müntener 1 , Jérémy Kottelat 1 , Annika Huber 1 , Daniel Häussinger 1
Affiliation  

Lanthanide chelating tags (LCTs) have been used with great success for determining structures and interactions of proteins and other biological macromolecules. Recently LCTs have also been used for in-cell NMR spectroscopy, but the bottleneck especially for demanding applications like pseudocontact shift (PCS) NMR is the sparse availability of suitable tags that allow for site-selective, rigid, irreversible, fast, and quantitative conjugation of chelated paramagnetic lanthanide ions to proteins via reduction stable bonds. We report here several such tags and focus on a new pyridine thiazole derivate of DOTA, that combines high affinity, rigidity, and selectivity with unprecedented tagging properties. The conjugation to the cysteine thiol of the protein results in a reductively stable thioether bond and proceeds virtually quantitatively in less than 30 min at 100 μM protein concentration, ambient temperature, and neutral pH. Upon conjugation of the new tag to two single cysteine mutants of ubiquitin and a single cysteine mutant of human carbonic anhydrase type II (30 kDa) only one stereoisomer is formed (square antiprismatic coordination, Λ(δδδδ)) and large to very large pseudocontact shifts as well as large residual dipolar couplings (RDCs) are observed by NMR spectroscopy. The PCS and RDC show excellent agreement with the solid state structure of the proteins. We believe that the pyridine thiazole moiety reported here has the potential to serve as a thiole reactive group in various conjugation applications; furthermore, its terbium complex shows strong photoluminescence upon irradiation and may thus serve as a donor group for Förster resonance energy transfer spectroscopy.

中文翻译:

用于PCS NMR光谱的新型镧系元素螯合标签,具有还原稳定的刚性连接基,可快速不可逆地偶联至蛋白质

镧系元素螯合标签(LCT)已成功用于确定蛋白质和其他生物大分子的结构和相互作用。最近,LCT也已用于细胞内NMR光谱学,但瓶颈尤其是对于要求苛刻的应用(如伪接触位移(PCS)NMR)而言,合适的标签稀少,可以通过还原稳定地将螯合的顺磁性镧系元素离子与蛋白质进行位点选择性,刚性,不可逆,快速和定量的缀合债券。我们在此报告了几种此类标签,并着重介绍了DOTA的新型吡啶噻唑衍生物,该衍生物结合了高亲和力,刚性和选择性以及前所未有的标签特性。与蛋白质的半胱氨酸硫醇的缀合导致还原稳定的硫醚键,并且在100μM蛋白质浓度,环境温度和中性pH值下,在不到30分钟的时间内几乎定量地进行。在将新标签缀合到两个泛素的单个半胱氨酸突变体和人类II型碳酸酐酶(30 kDa)的单个半胱氨酸突变体上时,仅形成一个立体异构体(方形反棱镜配位,Λ(δδδδ))和大到非常大的伪接触位移NMR光谱观察到大的残留偶极偶合(RDC)。PCS和RDC与蛋白质的固态结构显示出极好的一致性。我们认为,本文报道的吡啶并噻唑部分具有在各种偶联应用中充当硫醇反应性基团的潜力。此外,其complex络合物在辐照时显示出强的光致发光,因此可以用作福斯特共振能量转移光谱的供体基团。通过NMR光谱观察到Λ(δδδδ))和大到非常大的伪接触位移以及大的残留偶极耦合(RDC)。PCS和RDC与蛋白质的固态结构显示出极好的一致性。我们认为,本文报道的吡啶并噻唑部分具有在各种偶联应用中充当硫醇反应性基团的潜力。此外,其complex络合物在辐照时显示出强的光致发光,因此可以用作福斯特共振能量转移光谱的供体基团。通过NMR光谱观察到Λ(δδδδ))和大到非常大的伪接触位移以及大的残留偶极耦合(RDC)。PCS和RDC与蛋白质的固态结构显示出极好的一致性。我们认为,本文报道的吡啶并噻唑部分具有在各种偶联应用中充当硫醇反应性基团的潜力。此外,其complex络合物在辐照时显示出强的光致发光,因此可以用作福斯特共振能量转移光谱的供体基团。我们认为,本文报道的吡啶并噻唑部分具有在各种偶联应用中充当硫醇反应性基团的潜力。此外,其complex配合物在辐照下显示出强的光致发光,因此可以用作福斯特共振能量转移光谱的供体基团。我们认为,本文报道的吡啶并噻唑部分具有在各种偶联应用中充当硫醇反应性基团的潜力。此外,其complex配合物在辐照下显示出强的光致发光,因此可以用作福斯特共振能量转移光谱的供体基团。
更新日期:2018-08-30
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