The application of 199Hg NMR and 199mHg perturbed angular correlation (PAC) spectroscopy to define the biological chemistry of HgII:: A case study with designed two- and three-stranded coiled coils

The use of de novo designed peptides is a powerful strategy to elucidate Hg-II-protein interactions and to gain insight into the chemistry of Hg-II in biological systems. Cysteine derivatives of the designed a-helical peptides of the TRI family [Ac-G(L K(b)A(c)L(d)E(e)E(f)K(g))(4)-G-NH2] bind Hg-II at high pH values and at peptide/Hg-II ratios of 3:1 with an unusual trigonal thiolate coordination mode. The resulting Hg-II complexes are good water-soluble models for Hg-II binding to the protein MerR. We have carried out a parallel study using Hg-199 NMR and Hg-199m perturbed angular correlation (PAC) spectroscopy to characterize the distinct species that are generated under different pH conditions and peptide TRI L9C/Hg-II ratios. These studies prove for the first time the formation of [Hg{(TRI L9C)(2)-(TRI L9C-H)}], a dithiolate-Hg-II complex in the hydrophobic interior of the three-stranded coiled coil (TRI L9C)(3) Hg-199 NMR and Hg-199m PAC data demonstrate that this dithiolate-Hg" complex is different from the dithiolate [Hg(TRI L9C)(2)], and that the presence of third alpha-helix, containing a protonated cysteine, breaks the symmetry of the coordination environment present in the complex [Hg(TRI L9C)(2)]. As the pH is raised, the deprotonation of this third cysteine generates the trigonal thiolate-Hg-II complex Hg(TRI L9C)(3)(-) on a timescale that is slower than the NMR timescale (0.01-10 ms). The formation of the species {(TRI L9C)(2) (TRI L9C-H)}] is the result of a compromise between the high affinity of Hg-II to form dithiolate complexes and the preference of the peptide to form a three-stranded coiled coil.