De Novo Design of a Single Chain Diphenylporphyrin Metalloprotein

We describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV-vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization, and introduction of spectroscopic probes

Dynamics of the Globular Cluster System Associated with M87 (NGC 4486). II. Analysis

We present a dynamical analysis of the globular cluster system associated with M87 (= NGC 4486), the cD galaxy near the dynamical center of the Virgo cluster. The analysis utilizes a new spectroscopic and photometric database which is described in a companion paper (Hanes et al. 2001). Using a sample of 278 globular clusters with measured radial velocities and metallicities, and new surface density profiles based on wide-field Washington photometry, we study the dynamics of the M87 globular cluster system both globally --- for the entire cluster sample --- and separately --- for the metal-rich and metal-poor globular cluster samples. This constitutes the largest sample of radial velocities for pure Population II tracers yet assembled for any galaxy. We discuss the implications of our findings for models for the formation of giant elliptical galaxies, globular cluster systems, and the Virgo cluster. (ABRIDGED)Comment: 28 pages, 19 postscript figures, 1 jpeg image. See http://www.physics.rutgers.edu/ast/ast-rap.html to download the manuscript with higher quality figures. Accepted for publication in the Astrophysical Journa

Using α-Helical Coiled-Coils to Design Nanostructured Metalloporphyrin Arrays

We have developed a computational design strategy based on the alpha-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors

De novo design and characterization of a family of metalloporphyrin proteins: The porphyrin assemblers

De novo protein design utilizes computational and rational methods to produce proteins from pure principles. This approach critically tests our understanding of the basic principles underlying protein structure and function, while also enabling the design of structures with desirable properties that are not found in natural proteins. However, until now, de novo designed has been used primarily to reengineer proteins with structures and functions that closely resemble those found in natural proteins. We therefore explored computational and rational approaches to design and construct an asymmetric single chain protein bundle that self assembles and non-covalently binds a non-biological cofactor. Specifically incorporated into the scaffolds were interactions important for the function and stability of the bundles, including bis-His ligation of the non-biological cofactor, second shell hydrogen bonds, and favorable interactions between the cofactor and surrounding residues. Presented in this thesis are proteins that specifically recognize non-natural optically and electrically active cofactors. Briefly, Chapter Two focuses on the computational design of a protein that strongly resembles a natural four-helix bundle protein, but that recognizes a novel synthetic metalloporphyrin cofactor, the prototype protein, PAsc. PAsc was found to be monomeric, helical, and well folded, as demonstrated by size exclusion chromatography, AUC, CD, and NMR. PAsc also exhibits increased stability and helicity upon addition of this non-biological cofactor unique fold. Furthermore, computational and rational techniques were used to also change the prototype scaffold to examine the coordination of different metal cofactors and diversify the protein\u27s exterior, which is described in Chapters Four and Five. In a second application in Chapter Three, I demonstrate how the computational approach can be expanded to enable the design of a more novel protein-like scaffold that assembles porphyrins into linear arrays. The techniques used in Chapter Two are used here to demonstrate these arrays are tetrameric, well folded, and assemble around their designed number of cofactors. Prior to the work presented within, sequence-based design techniques had been used to design peptides that bind biological cofactors non-selectively. The design method provides control over the number, spacing, and chemical structure of the porphyrins, suggesting possible applications in the area of smart materials and nanotechnology