Paramagnetic Ligand Tagging To Identify Protein Binding Sites.
Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein-ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein-ligand systems with varying binding affinity.
Department of Chemistry and Molecular Biology and the Swedish NMR Centre, University of Gothenburg , SE-412 96 Gothenburg, Sweden.
Ulrika
U
Brath
Department of Chemistry and Molecular Biology and the Swedish NMR Centre, University of Gothenburg , SE-412 96 Gothenburg, Sweden.
Shashikala I
SI
Swamy
Department of Chemistry and Molecular Biology and the Swedish NMR Centre, University of Gothenburg , SE-412 96 Gothenburg, Sweden.
Alberte X
AX
Veiga
Department of Biochemistry and Molecular Biology, University of British Columbia , Vancouver, BC V6T 1Z3, Canada.
Ching-Chieh
CC
Tung
Department of Biochemistry and Molecular Biology, University of British Columbia , Vancouver, BC V6T 1Z3, Canada.
Filip
F
Van Petegem
Department of Chemistry and Molecular Biology and the Swedish NMR Centre, University of Gothenburg , SE-412 96 Gothenburg, Sweden.
Máté
M
Erdélyi
D000777:Anesthetics / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008024:Ligands; D060328:Magnetic Phenomena; D009682:Magnetic Resonance Spectroscopy; D008738:Methyl Ethers / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D000077149:Sevoflurane
D000777:Anesthetics; D002147:Calmodulin; D028581:Lanthanoid Series Elements; D008024:Ligands; D008738:Methyl Ethers; D011506:Proteins; D000077149:Sevoflurane