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 the Tri family. The Tri peptides are based on the parent sequence Ac-NH-G(LKALEEK)(x)()G-CONH(2) 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 leucines in the other a and d positions; Baby L9C (x = 3), which also has a cysteine 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 coiled 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 observed to be highly helical in the absence of denaturant at pH 8.5 while Baby L9C contained <20% helical content at pH 8.5, indicating a weakly associated or unassociated coiled coil. Size-exclusion chromatography (SEC) verified that Baby L9C was a monomer at pH 8.5. The helicity of Baby L9C was induced by addition of HgCl(2). 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 HgS(3) UV band at 248 nm. Titrations of Tri L9C and Baby L9C into solutions of HgCl(2) at pH values between 7 and 9 were performed to extract binding constants. Global fits to the data employed a mechanism that involved initial binding of mercury to the peptides forming a two-stranded coiled coil with linear thiolato Hg(II) at [peptide]/[Hg] <2, followed by addition of a more weakly associated third helix to generate a three-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 coil by SEC in a solution of Tri L9C at pH 7.0.
Farrer
B T
BT
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Harris
N P
NP
Balchus
K E
KE
Pecoraro
V L
VL
D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D008628:Mercury / Q000737:chemistry* / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013057:Spectrum Analysis; D013816:Thermodynamics
D008667:Metalloproteins; D010446:Peptide Fragments; D008628:Mercury; D019791:Guanidine