Thermodynamic model for the stabilization of trigonal thiolato mercury(II)in designed three-stranded coiled coils

A thermodynamic model is presented that describes the binding of Hg(II) to de novo designed peptides, Tri L9C and Baby L9C, which were derived from th e Tri family. The Tri peptides are based on the parent sequence Ac-NH-G(LKA LEEK)(x)G-CONH2 and are known to form two-stranded coiled coils at low pH ( pH <4) and three-stranded coiled coils at high pH (pH >7). Tri L9C (x = 4) contains a four heptad repeat sequence with cysteine in position 9 and leuc ines in the other a and d positions; Baby L9C (x = 3), which also has a cys teine in position 9 but is one heptad shorter than Tri L9C, was designed to form less stable helical coiled coils in solution. The free energies of co iled coil formation for Tri, Tri L9C. Baby Tri, and Baby L9C at pH 2.5 and 8.5 were determined by guanidinium denaturation titrations; Tri L9C was obs erved to be highly helical in the absence of denaturant at pH 8.5 while Bab y L9C contained < 20% helical content at pH 8.5, indicating a weakly associ ated or unassociated coiled coil. Size-exclusion chromatography (SEC) verif ied that Baby L9C was a monomer at pH 8.5. The helicity of Baby L9C was ind uced by addition of HgCl2. The subsequent formation of a trigonal thiolato Hg(II) in the interior of a three-stranded coiled coil was verified by the presence of a characteristic HgS3 UV band at 248 nm. Titrations of Tri L9C and Baby L9C into solutions of HgCl2 at pH values between 7 and 9 were perf ormed to extract binding constants. Global fits to the data employed a mech anism that involved initial binding of mercury to the peptides forming a tw o-stranded coiled coil with linear thiolato Hg(II) at [peptide]/[Hg] <2, fo llowed by addition of a more weakly associated third helix to generate a th ree-stranded coiled coil. This mechanism would require the deprotonation of the third cysteine thiol to generate the trigonal thiolato Hg(II) at pH >7 .5 [the pK(a) of the cysteine thiol in the presence of Hg(II)]. Support for this mechanism was given by the observation of a three-stranded coiled coi l by SEC in a solution of Tri L9C at pH 7.0.