Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Bioconjug Chem. 2013 Feb 20;24(2):260-8. doi: 10.1021/bc300631z. Epub 2013 Jan 16.
Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide-alkyne cycloaddition to a genetically encoded p-azido-l-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.
假接触位移 (PCS) 来自顺磁镧系离子，通过 NMR 光谱为结构生物学提供强大的远程结构约束，但蛋白质的镧系元素特异性标记仍然是一个挑战，因为大多数可用的镧系元素标记需要带有单个半胱氨酸残基的蛋白质。我们表明，通过铜 (I) 催化的叠氮化物-炔烃环加成反应，将基于环烯的顺磁镧系元素标签以位点特异性方式连接到蛋白质上，与带有连接物的遗传编码的 p-叠氮基-l-苯丙氨酸残基连接，该连接物足够短且刚性，可在几种蛋白质中观察到 PCS。尽管与体积庞大的标签相关的空间要求以及靠近蛋白质表面的反应条件苛刻，但在 Cu(I)-稳定配体 BTTAA 的帮助下，连接产物的产率始终保持在 50%以上，接近 100%。无论是否存在半胱氨酸残基，产率都很高，从而避免了与传统镧系元素标记策略相关的半胱氨酸突变的需要。
