A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual (15)N,(1)H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.
In this paper we show that water insoluble compounds dissolved in poly-gamma-benzyl-glutamate are amenable to the measurement of a number of homo- and heteronuclear dipolar couplings. The sensitivity and experimental precision of dipolar couplings are sufficient to obtain a good match with the structure. In order to achieve the necessary precision for H,C dipolar couplings between protons and carbons that are not directly bound a new method for the measurement of heteronuclear long range couplings is introduced that allows a one-parameter fit to a HSQC-based experiment as reference experiment. The methodology is applied to menthol (1R, 3S, 4R).
We describe the synthetic route to ethylenediaminetetraacetic acid (EDTA) derivatives that can be attached to surface-exposed thiol functional groups of cysteine residues in proteins, via a methylthiosulfonate moiety that is connected in a stereochemically unique way to the C-1 carbon atom of EDTA. Such compounds can be used to align proteins in solution without the need to add liquid crystalline media, and are, therefore, of great interest for the NMR spectroscopic analysis of biomolecules. The binding constant for the paramagnetic tag to lanthanide ions was determined by measuring luminescence. For the Tb(+3)-ligand complex, a K(b) value of 6.5 x 10(17) M(-1) was obtained. This value is in excellent agreement with literature values for the related EDTA compound. In addition, it could be shown that there is no significant reduction in the luminescence intensity upon addition of a 10(4) excess of Ca2+ ions, indicating that this paramagnetic tag is compatible with buffers containing high concentrations of divalent alkaline earth ions.
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