Completion of an Undulator-Based X-ray Scattering Facility for Materials Research on Complex Fluids

A synchrotron radiation-based X-ray scattering facility for materials research on complex fluids has been completed on Sector 09 at the Advanced Photon Source at Argonne National Laboratory. It consists of a beamline on an undulator magnet source with doubly focusing X-ray optics and endstation spectrometers for both small angle X-ray scattering and liquid surface X-ray scattering

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

Computational De Novo Design and Characterization of a Protein That Selectively Binds a Highly Hyperpolarizable Abiological Chromophore

This work reports the first example of a single-chain protein computationally designed to contain four α-helical segments and fold to form a four-helix bundle encapsulating a supramolecular abiological chromophore that possesses exceptional nonlinear optical properties. The 109-residue protein, designated SCRPZ-1, binds and disperses an insoluble hyperpolarizable chromophore, ruthenium(II) [5-(4\u27-ethynyl-(2,2\u27;6\u27,2″-terpyridinyl))-10,20-bis(phenyl)porphinato]zinc(II)-(2,2\u27;6\u27,2″-terpyridine)(2+) (RuPZn) in aqueous buffer solution at a 1:1 stoichiometry. A 1:1 binding stoichiometry of the holoprotein is supported by electronic absorption and circular dichroism spectra, as well as equilibrium analytical ultracentrifugation and size exclusion chromatography. SCRPZ-1 readily dimerizes at micromolar concentrations, and an empirical redesign of the protein exterior produced a stable monomeric protein, SCRPZ-2, that also displayed a 1:1 protein:cofactor stoichiometry. For both proteins in aqueous buffer, the encapsulated cofactor displays photophysical properties resembling those exhibited by the dilute RuPZn cofactor in organic solvent: femtosecond, nanosecond, and microsecond time scale pump-probe transient absorption spectroscopic data evince intensely absorbing holoprotein excited states having large spectral bandwidth that penetrate deep in the near-infrared energy regime; the holoprotein electronically excited triplet state exhibits a microsecond time scale lifetime characteristic of the RuPZn chromophore. Hyper-Rayleigh light scattering measurements carried out at an incident irradiation wavelength of 1340 nm for these holoproteins demonstrate an exceptional dynamic hyperpolarizabilty (β1340 = 3100 × 10(-30) esu). X-ray reflectivity measurements establish that this de novo-designed hyperpolarizable protein can be covalently attached with high surface density to a silicon surface without loss of the cofactor, indicating that these assemblies provide a new approach to bioinspired materials that have unique electro-optic functionality

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

Planar liquid-like arrangement of photopigment molecules in frog retinal receptor disk membranes

Low-angle X-ray diffraction arising from 40 to 50 A particles within wet frog retinal receptor disk membranes at 26 [deg]C was not consistent with a planar crystalline lattice of the particles within the disk membranes. The nature of the diffraction suggested the possibility of a planar liquid-like arrangement of the particles. Such an arrangement is supported by the observation that the planar ordering of the particles is easily altered by their interaction with globular protein molecules non-specifically adsorbed to the disk membranes. In view of the above, we obtained diffraction patterns from our wet disk membrane preparations at several temperatures between 4.5 and 42.5 [deg]C, and applied a Fourier analysis to the diffracted intensities appropriate for a planar liquid-like arrangement of the 40 to 50 A particles. The analysis gave the planar radial distribution function description of the supposed planar liquid-like arrangement of the particles. These radial distribution functions, derived from the diffracted intensities, were examined in terms of their shape and variation with temperature, and compared with the known predictions from statistical mechanics for a liquidlike arrangement of particles whose pair potential contains both attractive and repulsive terms. This comparison for the derived radial distribution functions demonstrated that the observed diffraction data from the 40 to 50 A particles were indeed consistent with a planar liquid-like arrangement of these particles within the disk membrane.Our radial distribution function analysis allowed model scattering factors for the particles to be tested. It was found that only hard sphere cross-sectional electron densities for the particle with diameters of 40 to 44 A or reasonably hard, soft-sphere cross-sectional electron densities, with a core of uniform electron density 38 to 40 A in diameter and a total diameter of 44 to 46 A, gave good agreement.A similar analysis was applied to the diffracted intensities arising from the antirhodopsin molecules adsorbed to the wet disk membranes which had been treated with our antirhodopsin serum and is discussed relative to the preceding paper (Blasie, Worthington & Dewey, 1969). A comparison of the radial distribution functions for the antirhodopsin molecules adsorbed to the antirhodopsin serum treated disk membranes and the 40 to 50 A particles of the untreated disk membranes at identical temperatures showed the particles to be the photopigment molecules.The mathematical derivation of the planar radial distribution function and a critical evaluation of the errors involved are presented in the Appendices.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33022/1/0000406.pd

Molecular localization of frog retinal receptor photopigment by electron microscopy and low-angle X-ray diffraction

Low-angle X-ray diffraction patterns were obtained from ordered ultracentrifugal pellets of wet receptor disk membranes which had been either treated with antirhodopsin serum, normal rabbit serum, serum albumin, or untreated prior to sedimentation. A preliminary analysis of these patterns indicated: (a) differences between antirhodopsin serum treated and untreated preparations are due to conjugation of antirhodopsin molecules with their antigen in the disk membranes and not non-specific adsorption of other serum proteins to the disk membranes, (b) the planar ordering of the adsorbed antirhodopsin molecules over the surface of the disk membrane is nearly identical to that of the 40 to 50 A particles in the untreated disk membrane.A detailed Fourier analysis of these patterns in terms of a planar liquid-like arrangement of the 40 to 50 A particles in the untreated disk membranes and the antirhodopsin molecules adsorbed to the antirhodopsin serum treated disk membranes confirmed our preliminary analysis. The planar liquid-like arrangement of the 40 to 50 A particles is nearly identical to that of the adsorbed antirhodopsin molecules (3.0 and 3.1 nearest neighbors at a separation of 56 and 58 A respectively at 26 [deg] +/- 0.2 deg.C). Thus, the 40 to 50 A particles of the wet untreated disk membranes are the photopigment molecules.Electron micrographs of phosphotungstate negatively-stained disk membrane demonstrate particles ~40 A in diameter within the disk membrane. Optical transforms of these electron micrographs show that these particles appearing in the micrograph are arranged in a planar square array with a unit cell side of ~ 70 A. Correlation of these results with those obtained by low-angle X-ray diffraction in ultracentrifugal pellets of phosphotungstate stained and dried disk membranes as well as on wet pellets of untreated disk membranes before, during and after drying indicates the following: the ~ 40 A diameter particles seen in the electron micrographs are most likely the same 40 to 50 A particles giving rise to the observed low-angle X-ray diffraction from wet disk membranes. Hence the particles seen in the electron micrographs are most likely the nonpolar cores of the photopigment molecules.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33021/1/0000405.pd