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Characterization of caged compounds binding to proteins by NMR spectroscopy |
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| Authors: | Joanna Bandorowicz-Pikula,Jesus Jimé nez-Barbero,Agnieszka Strzelecka-Kiliszek |
| Affiliation: | a Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02093 Warsaw, Poland b ICBMS UMR CNRS 5246, Université Lyon I, 69622 Villeurbanne Cedex, France c Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain d Anabio, Université Lyon 1, CNRS UMR 5180, Sciences Analytiques, 69622 Villeurbanne Cedex, France e Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. Da República, 2780-157 Oeiras, Portugal f Warsaw University of Life Sciences, SGGW, Nowoursynowska 166, 02787 Warsaw, Poland |
| Abstract: | Photolysable caged ligands are used to investigate protein function and activity. Here, we investigate the binding properties of caged nucleotides and their photo released products to well established but evolutionary and structurally unrelated nucleotide-binding proteins, rabbit muscle creatine kinase (RMCK) and human annexin A6 (hAnxA6), using saturation transfer difference NMR spectroscopy. We detect the binding of the caged nucleotides and discuss the general implications on interpreting data collected with photolysable caged ligands using different techniques. Strategies to avoid non-specific binding of caged compound to certain proteins are also suggested. |
| Keywords: | Anx, annexin CK, creatine kinase DMNPE-caged GTP-γ-S, guanosine 5&prime -O-(3-thiotriphosphate), P 3(S)-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester, triammonium salt NPE-ADP, adenosine 5&prime -diphosphate, P2-(1-(2-nitrophenyl)ethyl) ester, disodium salt RIDS, reaction-induced infrared difference spectroscopy RMCK, rabbit muscle creatine kinase STD, saturation transfer difference spectroscopy |
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