首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Site-specific labeling of proteins with cyclen-bound transition metal ions
Authors:Allison LewinJonathen P Hill  Ruth BoetzelTheoni Georgiou  Richard James  Colin KleanthousGeoffrey R Moore
Institution:a Centre for Metall. Spect. and Biology, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, UK
b School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
Abstract:A general procedure for site-specific and reversible labeling of proteins with transition metal ions is described. The method is based on the use of the novel ligand 1-(2-thioethyl)-1,4,7,10-tetraazacyclododecane (TETAC), which specifically and readily reacts with thiol groups. Synthesis of TETAC from 1,4,7,10-tetraazacyclododecane (cyclen) and ethylene disulfide yielded a mixture of products, including TETAC and its oxidized disulfide in 56.4% yield. The procedure for labeling proteins with TETAC is straightforward and led to separation of the TETAC-containing product mixture through gel-filtration chromatography. The resulting protein-TETAC adducts were shown to contain a single TETAC group which bound transition metal ions. Protein-TETACCu2+ had a UV-Vis spectrum similar to that of Cu2+(cyclen) while the protein-TETACCo2+ complex had a different spectrum to that of the cobalt-containing cyclen. This is because attachment to the protein prevented the Co2+-containing TETAC from dimerising and binding O2, which the cobalt-containing cyclen is able to do. The proteins used to develop this labeling procedure were the DNase domain of colicin E9 and its inhibitor protein Im9. Unlike Im9, the DNase does not contain a cysteine residue but the Ser30Cys variant of the DNase was prepared by site-directed mutagenesis. Both Im9 and the Ser30Cys DNase were modified with TETAC and the modifications shown to be structurally and functionally benign through NMR spectroscopy of the modified Im9 and fluorescence spectroscopy binding assays in which DNase-Im9 complexes were formed. The simplicity of the method, and its general application to any protein through the introduction of cysteine by site-directed mutagenesis, suggests it will be of wide use in protein chemistry applications.
Keywords:1-(2-Thioethyl)-1  4  7  10-tetraazacyclododecane  Labeling proteins  Colicin  Spectroscopy
本文献已被 ScienceDirect 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号