Designed Metalloproteins: From Structurally Characterized Scaffolds to Helical Bundles.

Fundamental studies using two classes of de novo designed alpha-helical scaffolds (three-stranded coiled coils or a single chain three-helix bundle), spectroscopy, and X-ray crystallography are exploited to enhance our understanding of the biochemistry of heavy metals Cd(II), Hg(II), and Pb(II) in thiol-rich de novo designed peptides. The X-ray structure of a parallel three-stranded coiled coil (CoilSer L9C) is determined to 1.36 Å resolution in its non-metallated form (Rw= 18.2%; Rf= 21.5%). Cys side chains show alternate conformations and are partially (15%) preorganized for metal binding at the polypeptide interior. How the physical properties of two Cd(II) ions bound in “identical” first coordination spheres at different topological positions within the same three-stranded coiled coil peptide vary has been investigated. GrandL16PenL19IL23PenL26I sequesters two Cd(II) ions in trigonal planar CdS3 geometries with similar physical properties; however, these two sites are not independent from each other. Metallation state (apo or [Cd(pep)(Hpep)2]+ or [Cd(pep)3]-) of one site perturbs the physical properties of the second site. GrandL12AL16CL26AL30C binds two Cd(II) ions as 4-coordinate CdS3O (O = water molecule) at both the binding sites, but with dramatically different properties. The 4-coordinate sites display differences in thermodynamic (pKa2 for Cd(II) binding ranging from 9.9 to 13.9) and kinetic (L26AL30C is more dynamic than L12AL16C site) properties depending on their location. Exchange studies have been performed to understand the mechanism of insertion of Cd(II) into and out of helical scaffolds. A multi-site exchange scheme is invoked. Free Cd(II) is believed to interact with surface Glutamates before being bound within the peptide interior. The process occurs on the slow exchange regime with rates ranging from 5.8 to 10.9 ms. CdS3 sites do not exchange on the NMR time scale. A tris(cysteine) metal binding site was engineered at the C-terminal end of an existing de novo designed three-helix bundle yielding alpha3DIV, a well folded and stable protein capable of binding heavy metals with high affinity (>10^7 M-1). Hg(II) is complexed as a linear HgS2 (low pH) / trigonal planar HgS3 complex (high pH), Cd(II) as four coordinate CdS3O/CdS3N (N = His72), and Pb(II) as trigonal pyramidal PbS3 species.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/84478/1/saumen_1.pd