Heteronuclear filters for two-dimensional proton NMR. Identification of the metal-bound amino acids in metallothionein and observation of small heteronuclear long-range couplings

NMR Spectroscopy of Paramagnetic Metalloproteins

“…[65] 2.5. Simultaneous use of paramagnetism-based restraints As already pointed out, all the paramagnetism-based restraints (d pcs , R 1para , paramagnetic ccrs, self-orientation rdcs) have been experimentally demonstrated to be consistent with one another and with the NOEs, the dihedral angles based on 3 J values, and the other diamagnetic restraints. The protein calbindin D 9k has been used to demonstrate the simultaneous use of all of them.…”

“…The k 2 value is usually negligible with respect to the k 1 value in the case of R 1 data but not in the case of R 2 data (see below). Equation (3) shows that the relaxation rate enhancement effects depend on the inverse of the 6th power of the metal-tonucleus distance, thus they tend to vanish rapidly. As anticipated in Section 1, they are measurable in a spherical shell from the metal (Figure 2) where the effect is not too weak to be negligible and not too strong to make the signals unobservable.…”

“…In some lucky cases, metal-nucleus-proton scalar connectivities can be exploited to prove the existence of a metal-protein bond. This is the case with 113 Cd [3][4][5][6] and 199 Hg, [7,8] for which metal-ligand couplings can be detected by heteronuclear 2D experiments such as metal-proton correlation or metal-edited spectra. These experiments provide crosspeaks originating from the metal-nucleus-proton couplings, which are directly related to the MXCH dihedral angle (M = metal ion; X = donor atom; Figure 1).…”

“…Finally, a section is dedicated to the significant progress made on 13 C direct detection, which reduces the negative effects of paramagnetism and may constitute a new chapter in the whole field of NMR spectroscopy. 113 CdÀSÀCÀH dihedral angle, q, and the metal-proton coupling constant, 3 J, in Cd 7 -metallothionein and Cd-rubredoxin, with the best-fit curve shown. [9] ChemBioChem larger than a proton) and have relaxation times varying from 10 À13 -10 À8 s depending on the atomic number of the metal and on the particular occupancy of the atomic orbitals.…”

Heteronuclear filters in two-dimensional [1H, 1H]-NMR spectroscopy: combined use with isotope labelling for studies of macromolecular conformation and intermolecular interactions

“…However, in turn one then has to consider that coherent artefacts from insufficient relaxation between successive scans can accumulate if each scan for the data set with effective 7r(X)-editing pulse is always immediately followed by the corresponding scan for the data set without effective editing pulse. It has been shown by Worgotter et al (1988) that these artefacts can be suppressed by repetition of the phase cycle with permuted order of the phase of the editing (7r/2)(X)-pulse. In the first half of this extended phase cycle, each scan for data set A is followed by the corresponding scan for data set B [equation (2)], while in the second half the scans for data set A are sampled after the corresponding scans for data set B.…”