title: A paramagnetic probe to localize residues next to carboxylates on protein surfaces.
abstract: It is shown that the paramagnetic properties of lanthanides can be exploited to obtain information on specific parts of a protein surface. Owing to the high affinity of coordinatively unsaturated lanthanide complexes for oxygen donors, carboxylate groups can be used as preferential targets for the interaction. The DO3A ligand is particularly useful in these studies, as it coordinates lanthanides in a heptadentate fashion, leaving two sites available for exogenous donors. A solution of a (15)N-labeled sample protein, calbindin D(9k) (75 residues), was titrated with up to 200% of Gd(III)-DO3A complex, and an inversion recovery (15)N-(1)H HSQC experiment was used to measure the paramagnetic contributions to the longitudinal relaxation rates of the amide protons. Relaxation data were used as distance constraints to estimate the number of interacting complexes and the occupancies of their binding sites. Four preferential interaction sites on the protein surface are found. Inspection of the various carboxylate side chains on the surface of the protein indicates that Gd(III)-DO3A interacts preferentially with carboxylate-rich regions, rather than with isolated carboxylates, suggesting the possibility of chelation of one Gd(III)-DO3A molecule by two carboxylate groups. Gd(III)-DO3A is thus a valuable semi-selective probe for clusters of negative charges on the protein surface.
authors.affiliation: Department of Chemistry IFM, University of Turin, Via P. Giuria 7, Italy.
authors.forename: Silvio
authors.initials: S
authors.lastname: Aime
authors.forename: Nicola
authors.initials: N
authors.lastname: D'Amelio
authors.forename: Marco
authors.initials: M
authors.lastname: Fragai
authors.forename: Yong-Min
authors.initials: YM
authors.lastname: Lee
authors.forename: Claudio
authors.initials: C
authors.lastname: Luchinat
authors.forename: Enzo
authors.initials: E
authors.lastname: Terreno
authors.forename: Gianni
authors.initials: G
authors.lastname: Valensin
mesh_terms: D000465:Algorithms; D000818:Animals; D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D005682:Gadolinium / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009942:Organometallic Compounds / Q000737:chemistry*; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism; D013499:Surface Properties
chemical_list: D064026:Calbindins; D002264:Carboxylic Acids; C469806:Gd(III)-DO3A complex; D008024:Ligands; D009942:Organometallic Compounds; D064030:S100 Calcium Binding Protein G; D005682:Gadolinium; D002118:Calcium