Design of a Three‐Helix Bundle Capable of Binding Heavy Metals in a Triscysteine Environment

Abstract

An important objective of de novo protein design is the preparation of metalloproteins, as many natural systems contain metals that play crucial roles for the function and/or structural integrity of the biopolymer. [1,2] Metalloproteins catalyze some of the most important processes in nature, from energy generation and transduction to complex chemical transformations. At the same time, metals in excess can be deleterious to cells, and some ions are purely toxic, with no known beneficial effects (e.g., Hg II or Pb II ). Ideally, we would hope to be able to use an approach based on first principles to create both known metallocenters and novel sites, which may lead to exciting new catalytic transformations. However, the design of novel metalloproteins is a challenging and complex task, especially if the aim is to prepare asymmetric metal environments.Numerous metalloprotein systems have been designed over the past 15 years, typically through the use of unassociated peptides that assemble into three-stranded coiled coils or helix-loop-helix motifs that form antiparallel fourstranded bundles. In terms of metal-ion binding, these systems have been functionalized with heme [3,4] and nonheme mononuclear [5] and binuclear centers. [6,7] It is often difficult to prepare nonsymmetrical metal sites through these strategies owing to the symmetry of the systems, which rely on homooligomerization. Thus, the preparation of a single polypeptide chain capable of controlling a metal-coordination environment is a key objective.Previously, we designed soft, thiol-rich metal-binding sites involving cysteine and/or penicillamine as the ligating amino acid residues into the interior of parallel, three-stranded ahelical coiled coils. [8,9] These systems have served as hallmarks for understanding the metallobiochemistry of different heavy metals, such as Cd II , Hg II , As III , and Pb II . [8][9][10][11] We have shown how to control the geometry and coordination number of metals such as Cd II and Hg II at the protein interior and how to fine-tune the physical properties of the metals, which led to site-selective molecular recognition of Cd II . [12][13][14] Although these homotrimeric assemblies have been very useful, the production of heterotrimeric systems in which metal environments could be fine-tuned controllably or a hydrogen bond could be introduced site-specifically has been elusive. [15] Therefore, we chose an alternative strategy to satisfy this objective and used a single polypeptide chain instead of multiple self-associating peptides.Existing designed heteromeric helical bundles and coiled coils show energetic preferences of several kcal mol À1 for the desired heteromeric versus homomeric assemblies. [16,17] However, the energy gap between a hetero-and homomeric assembly often depends critically on ionic strength, the pH value, and other environmental parameters. Moreover, the objective of many studies in de novo protein design is to make the metal ion adopt an energetically suboptimal coordination geometry, and the degree t...

Natural and Artificial Proteins Containing Cadmium

“…111m Cd PAC confirmed that one signal is the result of 4-coordinate CdS 3 O [77] but showed that the second species is best described by a four coordinate CdS 3 N (o o ¼ 0.17 rad/ns), where N corresponds to the His side chain towards the flexible C-terminus, which is in close proximity to the metal binding site (Figure 12) [77]. Definitive correlation of the 113 Cd NMR signals to these two different structures awaits further experimentation.…”

“…Titration data confirmed a binding ratio of one Cd(II) per single strand of a 3 DIV as well as a binding constant of 2.0 Â 10 7 M -1 [77]. 113 Cd NMR of 113 Cd(a 3 DIV) -has two resonances at 595 (major) and 583 (shoulder) ppm, both of which would be consistent with predominantly CdS 3 O sites [77].…”

“…A trigonal Cys site was prepared by introducing one Cys into each ahelix toward the C-terminal end of the bundle in a relatively hydrophobic environment (Table 1 and Figure 12). Cd(II) binding to this peptide, a 3 DIV, was monitored by the formation of the characteristic LMCT transition at 232 nm with a molar absorption coefficient of 18200 M -1 cm -1 at pH 8, consistent with three Cys bound to Cd(II) [77]. Titration data confirmed a binding ratio of one Cd(II) per single strand of a 3 DIV as well as a binding constant of 2.0 Â 10 7 M -1 [77].…”

“…Cd(II) binding to this peptide, a 3 DIV, was monitored by the formation of the characteristic LMCT transition at 232 nm with a molar absorption coefficient of 18200 M -1 cm -1 at pH 8, consistent with three Cys bound to Cd(II) [77]. Titration data confirmed a binding ratio of one Cd(II) per single strand of a 3 DIV as well as a binding constant of 2.0 Â 10 7 M -1 [77]. 113 Cd NMR of 113 Cd(a 3 DIV) -has two resonances at 595 (major) and 583 (shoulder) ppm, both of which would be consistent with predominantly CdS 3 O sites [77].…”

A De Novo Designed Metalloenzyme for the Hydration of CO2

“…[12] Previously, our lab incorporated a Cys 3 metal binding site near the C-terminus of α 3 D and showed that the resulting protein, α 3 DIV, binds Hg(II), Pb(II), and Cd(II) with high affinity in coordination geometries previously identified within the TRI family of 3SCCs. [15] Here, we report a new metalloenzyme, α 3 DH 3 , which contains a His 3 site that, upon binding Zn(II), catalyzes the hydration of CO 2 ( Figure 1). α 3 DH 3 differs from α 3 D in that three leucine residues were replaced with histidine residues (L18H, L28H, L67H), a histidine residue was replaced with valine (H72V) to ensure no competition for Zn(II) binding, and four extra residues were added to the end of the chain (GSGA) which improved expression yields (Table 1).…”