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.

Paramagnetic NMR study of Cu2+?IDA complex localization on a protein surface and its application to elucidate long distance information

“…Other paramagnetic metal chelate complexes, such as Mn 2þ -NTA and Gd 3þ -EDTA, did not bind specifically to the histidine residues for three proteins possessing a poly hisitidine tag; HR23B UbL, IRF4 DBD and the S5a UbL binding region [18]. These results are consistent with hard-soft-acid-base (HSAB) theory [25][26][27].…”

Long‐Range‐Distance NMR Effects in a Protein Labeled with a Lanthanide–DOTA Chelate

“…Paramagnetic effects can be used in protein NMR analysis by addition of transition metals, [1,2] lanthanide ions, [3,4] lanthanide chelates, [5][6][7][8][9][10] stable radicals, such as 2,2,6,6-tetra-A C H T U N G T R E N N U N G methylpiperidine-1-oxyl (TEMPO), [11][12][13][14][15][16] or dioxygen, [17] to decrease the signal overlap of the resonances observed, to probe the molecular surface of these large molecules, or to align macromolecules for residual dipolar-coupling measurements.…”

Probing the Water Coordination of Protein‐Targeted MRI Contrast Agents by Pulsed ENDOR Spectroscopy

“…Moreover, it has been reported that the hydration number can be different for a complex bound to a protein target than for the complex in aqueous solution. [11,12] To design high-relaxivity contrast agents, it is important to be able to account for the various parameters affecting nuclear relaxation. The magnetic-field dependence of relaxivity gives only indirect insight into the metal coordination sphere, since a variety of parameters can be responsible for relaxivity and the change in relaxivity upon protein binding.…”