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. 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 or Pb). 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 and nonheme mononuclear and binuclear centers. 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. These systems have served as hallmarks for understanding the metallobiochemistry of different heavy metals, such as Cd, Hg, As, and Pb. We have shown how to control the geometry and coordination number of metals such as Cd and Hg at the protein interior and how to fine-tune the physical properties of the metals, which led to site-selective molecular recognition of Cd. 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. 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 kcalmol 1 for the desired heteromeric versus homomeric assemblies. However, the energy gap between a heteroand 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 to which this strategy will be successful depends on the size of the energy gap between the desired heteromeric assembly and other homomeric or misfolded states. Also, even when heterooligomeric bundles have been used to successfully identify specific environmental effects that influence substrate binding or the reactivity of a metal-ion cofactor, the noncovalently assembled complexes have often been difficult to characterize structurally, possibly owing to small populations of alternatively assembled species. In this case, the inclusion of the active-site residues in a construct with linked helices greatly facilitated structural analysis and catalytic characterization. An attractive starting scaffold to meet our objectives is the de novo designed three-helix bundle a3D. The structure of this protein has been determined by NMR spectroscopy, and it has been proven that the helices are oriented in a counterclockwise topology. Although the a3D protein originated from a coiled coil, its helices were shortened to such an extent that it might be better considered as a globular protein whose repetitive structure makes each of the heptads very similar to one another (in the absence of end effects). The stability of a3D is similar to that of natural proteins. Thus, a3D should be tolerant to mutations, and this protein should serve as an excellent framework for the engineering of specific metalbinding sites. Additionally, with this protein scaffold, we can study the effect of the ligating residue located on the second [*] S. Chakraborty, Dr. J. Yudenfreund Kravitz, Prof. V. L. Pecoraro Department of Chemistry, University of Michigan Ann Arbor, MI 48109 (USA) Fax: (+1)734-936-7628 E-mail: vlpec@umich.edu
Read full abstract