Thermodynamic and kinetic investigation of heavy metal binding to de novo designed alpha -helical peptides.

Abstract

Binding of Hg(II) and Cd(II) to variants of the <italic>de novo</italic> designed TRI [Ac-G(LKALEEK)₄G-NH₂] peptides, were performed to investigate fundamentals governing the role of metals in biology. Metal-peptide associations were studied with UV-Vis, CD, Stopped-Flow, <super>113</super>Cd-NMR, <super> 199</super>Hg-NMR and Analytical Ultracentrifugation experiments. All the peptides sequestered Hg(II) and Cd(II) strongly, in an unorthodox trigonal environment. For binding of Hg(II) to the peptides, dissociation constants ranged from 2.4x10<super>-5</super> M for the smallest peptide up to 2.5x10<super>-9</super> M for the longest, GrandL9C, for binding of the third thiolate to linear Hg(II)(pep)₂. Binding of Hg(II) to GrandL9C displayed &sim;50% trigonal Hg(II) formation at nanomolar metal concentrations, similar to MerR. Approximately 11 kcal/mol of the Hg(II)(GrandL9C)₃<super>-</super> stability is due to peptide interactions, while 1-4 kcal/mol stabilization results from Hg(II)(RS)₂ binding the third thiolate. For Cd(II), dissociation constants ranged from 1.3x10<super>-6</super> M up to 8.3x10<super>-9</super> M showing similar nature of stable aggregate formation. A linear free energy study correlating self-association affinity of the peptides versus binding affinity to Hg(II) and Cd(II) showed that the fundamental driving force for trigonal metal geometry attainment was dependent on the degree of association of the polypeptide. Kinetic investigation of insertion of Hg(II) into the peptides showed a well-separated biphasic behavior for metal encapsulation corresponding to the correct formation of a metal inserted three-stranded coiled coil (fast phase, k=720.8 M<super>-1</super>sec<super>-1</super>) and the rearrangement of the improperly folded coiled coil to the properly folded structure (slow phase, k=0.02 sec<super>-1</super>). The results for Cd(II) displayed a different mechanistic pathway with a fast step (k=1.8x10<super>4</super> M<super>-1 </super>sec<super>-1</super>) of metal ion binding to thiolate ligands in the folded peptide, followed by a slow pH dependent equilibrium (k=0.1 sec<super> -1</super>) between water bound (Cd(II)S₃(OH₂)<super> -</super>) and unbound (Cd(II)S₃<super>-</super>) species. Attempts at generating heteromeric TRI peptides demonstrated that the coiled coils were far more robust than was previously expected. Replacement of one strand of a coiled coil with a strand from another peptide and binding to Hg(II) failed to realize a 100% yield of a heteromeric bundle. Results of this work provided model compounds for studying energetics of the metalloregulatory proteins, MerR and CadC and understanding of metal coordination preferences on mechanisms of metal binding.