The cluster glycoside effect.

Truncation and mutation of a poorly folded 39-residue peptide has produced 20-residue constructs that are >95% folded in water at physiological pH. These constructs optimize a novel fold, designated as the 'Trp-cage' motif, and are significantly more stable than any other miniprotein reported to date. Folding is cooperative and hydrophobically driven by the encapsulation of a Trp side chain in a sheath of Pro rings. As the smallest protein-like construct, Trp-cage miniproteins should provide a testing ground for both experimental studies and computational simulations of protein folding and unfolding pathways. Pro Trp interactions may be a particularly effective strategy for the a priori design of self-folding peptides.

Designing a 20-residue protein.

The design of artificial catalysts able to compete with the catalytic proficiency of enzymes is an intense subject of research. Non-covalent interactions are thought to be involved in several properties of enzymatic catalysis, notably (i) the confinement of the substrates and the active site within a catalytic pocket, (ii) the creation of a hydrophobic pocket in water, (iii) self-replication properties and (iv) allosteric properties. The origins of the enhanced rates and high catalytic selectivities associated with these properties are still a matter of debate. Stabilisation of the transition state and favourable conformations of the active site and the product(s) are probably part of the answer. We present here artificial catalysts and biomacromolecule hybrid catalysts which constitute good models towards the development of truly competitive artificial enzymes.

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Supramolecular catalysis. Part 2: artificial enzyme mimics

The construction of complex protein folds relies on the precise conversion of a linear polypeptide chain into a compact 3-dimensional structure. The interplay of forces that link sequence and folding is intricate and yet to be firmly elucidated. Examination of protein 3-dimensional structures suggests that complex tertiary folds and quaternary associations can be deconstructed into a limited number of secondary structural elements, such as strands, helices, and turns, which are assembled using loosely structured loops (Figure 1). The stability of a specific fold is determined by tertiary interactions between residues which are distant in sequence. De novo design of existing or novel protein folds demands a thorough understanding of the rules that underlie protein structure and stability.

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Design of folded peptides.

Metalloproteins perform a variety of functions in biological systems. The catalysis of several remarkable chemical transformations occurs at metal centers embedded in the active sites of metalloenzymes. Metalloproteins having carboxylate-bridged diiron cores comprise an important class, with functional versatility despite obvious structural similarities.1-7 The functions performed by these proteins and their significance have inspired a range of biomimetic studies, which in turn suggest possible chemical, biological, and pharmaceutical applications.1,8-11 The development of small molecule synthetic model compounds for non-heme dinuclear iron-based metalloproteins is a challenging field. In this article we review two decades of progress in the area. Table 1 lists metalloproteins that have now been identified to share the carboxylate-bridged diiron active site motif.3,4,6,7 Their various roles in nature are delineated. Some have been thoroughly investigated, and their X-ray structures are available. Examples include hemerythrin,12 methane monooxygenase,13,14 and ribonucleotide reductase.15,16 Others are assigned to the carboxylate-bridged diiron family according to gene sequence analysis and, occasionally, available spectroscopic data. The first carboxylate-bridged diiron protein to be discovered was hemerythrin (Hr), which has been studied since the 1950s.12 Hemerythrins are dioxygen carrier proteins found in marine invertebrates. They are functional analogues of the mammalian proteins myoglobin and hemoglobin. The active site of hemerythrin (Scheme 1) is composed of two iron atoms bridged by two carboxylate ligands from glutamate and aspartate residues. Five terminally bound histidine units and a bridging hydroxo group complete the coordination spheres of the two iron atoms, one of which is six-coordinate and the other five-coordinate in the reduced, diiron(II) state of deoxy Hr. Upon reaction with O2, deoxy Hr is converted to oxy Hr, in which an η1-hydroperoxo group binds to the available coordination site and forms a hydrogen bond to the bridging oxo unit. Biomimetic studies of this protein have advanced the understanding of the mechanism * To whom correspondence should be addressed. E-mail: lippard@ lippard.mit.edu. 987 Chem. Rev. 2004, 104, 987−1012

Synthetic models for non-heme carboxylate-bridged diiron metalloproteins: strategies and tactics.

De novo protein design has proven to be a powerful tool for understanding protein folding, structure, and function. In this Account, we highlight aspects of our research on the design of dimeric, four-helix bundles. Dimeric, four-helix bundles are found throughout nature, and the history of their design in our laboratory illustrates our hierarchic approach to protein design. This approach has been successfully applied to create a completely native-like protein. Structural and mutational analysis allowed us to explore the determinants of native protein structure. These determinants were then applied to the design of a dinuclear metal-binding protein that can now serve as a model for this important class of proteins.

De novo design of helical bundles as models for understanding protein folding and function.

De novo protein design provides an attractive means for testing and refining the principles governing the stability and tertiary structure of proteins. We describe the NMR solution structure of a 35-residue peptide designed to form a helix−loop−helix that dimerizes into a four-helix bundle. Structures were calculated on the basis of 834 NMR-derived restraints including 140 long-range NOEs. With 24 restraints per residue, the structure is well determined (0.28 A RMSD for backbone residues 3-33) and includes many features of the design yet adopts a novel topology that was unexpected. The forces that caused this peptide to adopt this unique fold are discussed.

R B Hill

Papers

De novo design of helical bundles as models for understanding protein folding and function.

Solution Structure of α2D, a Nativelike de Novo Designed Protein