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

Abstract

The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.

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

“…The attachment of the CLaNP-1 molecule to Paz in a bidentate manner was confirmed previously. [60] The { 15 N, 1 H} HSQC spectra of the Y CLaNP-1, the diamagnetic control probe, bound to this mutant exhibited only small chemical-shift perturbations for several residues in the neighborhood of the attachment site, relative to the wildtype Paz spectra, however the remaining amide resonances remained unaffected. The overall similarity between these two structures was also confirmed by X-ray crystallography results.…”

“…[65] Compound 1 (978 mg, 2.44 mmol) in CH 3 CN (100 mL) and dry K 2 CO 3 (4 g, 28.9 mmol) was treated dropwise with benzyl bromoacetate (0.8 mL, 5.0 mmol) under an argon atmosphere and stirring was continued for 48 h. The reaction mixture was filtered, concentrated, and purified by column chromatography (EtOAc:-MeOH 9:1 + 2 % TEA) to give compound 2 as a slightly yellow oil (480 mg, 28 %). 1 1,7-Bis(tert-butoxycarbonylmethylene)-1,4,7,10-tetraazacyclododecano-N''-N''''-bisA C H T U N G T R E N N U N G (2'-(acetylamino)ethylmethanesulfonothioate) 3 a: Compound 2 (300 mg, 0.45 mmol) in degassed ethanol (8 mL) was treated with 10 % palladium on carbon (16 mg) and then shaken under a hydrogen atmosphere (25 psi) for 48 h. The catalyst was removed with the aid of Hyflo, the filtrate was concentrated under reduced pressure, and traces of solvent were removed by using an oil pump. The residue was dissolved in chloroform (2 mL) by adding a small amount of DMF (0.05 mL) and the solution was added to a suspension of 2-(aminoethyl)methanethiosulfonate hydrobromide (222 mg, 0.94 mmol) in chloroform (2 mL) at 0 8C under an argon atmosphere in the presence of triethylamine (0.14 mL, 1.0 mmol) and HOBt (0.142 g, 1.05 mmol).…”

“…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.…”

“…[40] Although there are quite a few examples for which a paramagnetic metal exists or can be engineered inside the core of a protein, there are numerous others for which this is not possible. This disadvantage can be eliminated by the construction of paramagnetic probes Abstract: A two-thiol reactive lanthanide-DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) chelate, CLaNP-3 (CLaNP = caged lanthanide NMR probe), was synthesized for the rigid attachment to cysteine groups on a protein surface, and used to obtain long-range-distance information from the { 15 N, 1 H} HSQC spectra of the protein-lanthanide complex. The DOTA ring exhibits several isomers that are in exchange; however, single resonances were observed for most amide groups in the protein, allowing determination of a single, apparent magnetic-susceptibility tensor.…”

Ubiquitin Stability and the Lys 63‐Linked Polyubiquitination Site Are Compromised on Copper Binding

“…It is worth mentioning that in the case of the Cu II -iminodiacetic acid complex (Cu II -IDA), in which three coordination sites of Cu II were firmly occupied by the IDA ligand and the protein could provide only one easily accessible donor atom, Cu II coordination occurred to His 68 on the Ub surface. [17] In summary, we have located the primary binding of Cu II ions to a specific site at the N terminus of Ub, involving the Met 1 nitrogen and three oxygen donor ligands in a tetragonal geometry. Potential oxygen donor ligands are Met 1 and Val 17 (carbonyl groups), and Glu 16 and Glu 18 (carboxylate groups).…”