Design of three-dimensional domain-swapped dimers and fibrous oligomers.

Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 A resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.

Solution structure and dynamics of a de novo designed three-helix bundle protein.

Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25- to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-helix bundle protein, alpha3D, is reported. The structure of alpha3D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.

From coiled coils to small globular proteins: design of a native-like three-helix bundle.

A monomolecular native-like three-helix bundle has been designed in an iterative process, beginning with a peptide that noncooperatively assembled into an antiparallel three-helix bundle. Three versions of the protein were designed in which specific interactions were incrementally added. The hydrodynamic and spectroscopic properties of the proteins were examined by size exclusion chromatography, sedimentation equilibrium, fluorescence spectroscopy, and NMR. The thermodynamics of folding were evaluated by monitoring the thermal and guanidine-induced unfolding transitions using far UV circular dichroism spectroscopy. The attainment of a unique, native-like state was achieved through the introduction of: (1) helix capping interactions; (2) electrostatic interactions between partially exposed charged residues; (3) a diverse collection of apolar side chains within the hydrophobic core.

Expression of de novo designed alpha-helical bundles.

The successful design of proteins requires careful consideration of the multiplicity of forces that stabilize their three-dimensional structures including hydrophobic interactions, hydrogen-bonding, electrostatics and weakly polar interactions. Early attempts to design proteins relied too heavily on hydrophobic interactions to provide stability, resulting in structures with dynamic properties. Addition of more specific interactions to these initial designs gives rise to proteins with more native-like properties. This manuscript describes the design of native-like three- and four-helix bundles, and their cloning and expression of these proteins.

De novo design of heterotrimeric coiled coils.

The three-helix bundle is a common structural motif among natural proteins. It has been observed in numerous important proteins, such as fibrinogen, laminin, spectrin, dystrofin, hemagglutinin, and mannose binding proteins. The three-helix bundle is a simple structure in which three alpha-helices pack against each other, with a slight left-handed twist. Because of its simplicity relative to other structural motifs, the three-helix bundle can be conveniently used both to clarify the forces responsible for the protein folding and stability, and for the design of novel proteins. In this paper we describe the design, synthesis, and characterization of three peptides that self-assemble into antiparallel, heterotrimeric coiled coils. The experimental results, obtained from CD spectroscopy and ultracentrifugation equilibrium sedimentation, indicate that the mixture of the three peptides preferentially forms heterotrimers; moreover, these aggregates represent attractive systems for combinatorial design of libraries of pseudo C3 symmetric ligands or binding sites.

Protein design: a hierarchic approach.

The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of alpha-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.

Native-like and structurally characterized designed alpha-helical bundles.

A number of coiled coils and alpha-helical bundles have recently been designed, and many have now been structurally characterized by X-ray crystallography. Others have not been as well characterized structurally but exhibit native-like properties in aqueous solution. Both areas of investigation have contributed greatly to our understanding of the nature of specificity in this class of molecules.

Mercury-199 NMR of the metal receptor site in MerR and its protein-DNA complex.

Structural insights have been provided by mercury-199 nuclear magnetic resonance (NMR) into the metal receptor site of the MerR metalloregulatory protein alone and in a complex with the regulatory target, DNA. The one- and two-dimensional NMR data are consistent with a trigonal planar Hg-thiolate coordination environment consisting only of Cys side chains and resolve structural aspects of both metal ion recognition and the allosteric mechanism. These studies establish 199Hg NMR techniques as useful probes of the metal coordination environment of regulatory proteins, copper enzymes, and zinc transcription factor complexes as large as 50 kilodaltons.