The Application of199Hg NMR and199mHg Perturbed Angular Correlation (PAC) Spectroscopy to Define the Biological Chemistry of HgII: A Case Study with Designed Two- and Three-Stranded Coiled Coils

Abstract

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 alpha-helical peptides of the TRI family [Ac-G-(L(a)K(b)A(c)L(d)E(e)E(f)K(g))(4)-G-NH(2)] 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 (199)Hg NMR and (199m)Hg 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). (199)Hg NMR and (199m)Hg PAC data demonstrate that this dithiolate-Hg(II) 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 [Hg{(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.