Examining the Metal Selectivity of Thiol Rich de Novo Designed Peptides as a Strategy to Make Asymmetric Alpha-helical Protein Assemblies.

Abstract

The first preparation of metal-nucleated heterotrimers, asymmetric remote metal binding sites and CuI2 centers in de novo designed alpha-helical three-stranded coiled coils (3SCCs) is provided in this thesis. Peptides with Ala or Leu in the layer directly above a Cys¬3 site¬, [sequence: Ac-GLKALEEK LKAXEEK CKALEEK (LKALEEK)2G-NH2], with X=A (peptide A) and X=L (peptide B) were mixed as 2(A) and 1(B), with PbII to form, PbII(AAB) heterotrimers over the pH range 7.4 to 9.4 with a distinct 207Pb NMR chemical shift, (5600 ppm) compared to AAA (5575 ppm) and BBB PbII-homotrimers (5650 ppm). The coexistence of AAB and AAA chemical shifts in appropriate ratios confirms the formation of heterotrimers. The ability to make heterotrimeric 3SCCs removes previous restrictions due to the inherent self-association of the peptides, allowing for the design and analysis of asymmetric metal binding sites as observed in nature. AAB aggregates containing a second metal binding site with 0-3 Asp residues above a His3 site (AspXHis¬3) are described for potential catalytic applications. Prior to this thesis work, only symmetric metal binding sites could be achieved within the 3SCC scaffold. An Asp residue may be added to the Leu/Cys or the Ala/Cys containing helix. UV-Vis, EPR and XAS analysis of CoII-bound homotrimers reveal that 6-coordinate, highly symmetric CoII-homotrimers are formed whereas less symmetric, 5-coordinate environments result for the CoII-heterotrimers. The similarities between the heterotrimers and CoII-substituted carboxypeptidase A support direct CoII-Asp coordination in AAB systems. ZnII XAS analysis indicates ZnII is 4-coordinate in all ZnII-homo- and heterotrimers, demonstrating that CoII and ZnII coordinate differently. The spectroscopic evidence confirms the first inclusion of asymmetric metal binding sites within 3SCCs. Competition between CuI and PbII for Cys3 reveals unprecedented CuI2 sites. XAS of CuI-peptides are consistent with CuI-CuI back-scatterering (2.66 Å) and 2 or 3 CuI-S distances (2.20 Å). UV-Vis studies confirm a 2:1 binding stoichiometry. Mixtures of PbII and CuI yield CuIPbIICys3 proteins. Preformed CuI2Cys3 inhibits binding of PbII while addition of one equivalent of CuI to PbIICys3 does not displace PbII as CuICys3 features develop. Excess CuI is required to displace PbII, indicating comparable CuI and PbII binding affinities.