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...

Configurational entropy is thought to influence biomolecular processes, but there are still many open questions about this quantity, including its magnitude, its relationship to molecular structure, and the importance of correlation. The mutual information expansion (MIE) provides a novel and systematic approach to computing configurational entropy changes due to correlated motions from molecular simulations. Here, we present the first application of the MIE method to protein-ligand binding, using multiple molecular dynamics simulations (MMDSs) to study association of the UEV domain of the protein Tsg101 and an HIV-derived nonapeptide. The current investigation utilizes the second-order MIE approximation, which treats correlations between all pairs of degrees of freedom. The computed change in configurational entropy is large and is found to have a major contribution from changes in pairwise correlation. The results also reveal intricate structure-entropy relationships. Thus, the present analysis suggests that, in order for a model of binding to be accurate, it must include a careful accounting of configurational entropy changes.

Density functional theory has been used to investigate structural and electronic properties of complexes related to the resting form of the active site of vanadium haloperoxidase as a function of environment and protonation state. Results obtained by studying models of varying size and complexity highlight the influence of environment and protonation state on the structure and stability of the metal cofactor. The study shows that, in the trigonal bipyramidal active site, where one axial position is occupied by a key histidine, the trans position cannot contain a terminal oxo group. Further, a highly negatively charged vanadate unit is not stable. Protonation of at least one equatorial oxo ligand appears necessary to stabilize the metal cofactor. The study also indicates that, while at rest within the protein, the vanadate unit is most likely an anion with an axial hydroxide and an equatorial plane containing two oxos and a hydroxide. For the neutral, protonated state of the vanadate unit, there were two minima found. The first structure is characterized by an axial water with two oxo and one hydroxo group in the equatorial plane. The second structure contains an axial hydroxo group and an equatorial plane composed of one oxo and two hydroxo oxygen atoms. These two species are not significantly different in energy, indicating that either form may be important during the catalytic cycle. These data support the initial crystallographic assignment of an axially bound hydroxide, but an axial water is also a possibility. This study also shows that the protonation state of the vanadate ion is most likely greater than previously proposed.