Regina Valluzzi

Formation of silver and gold dendrimer nanocomposites

Structural types of dendrimer nanocomposites have been studied and the respective formation mechanisms have been described, with illustration of nanocomposites formed from poly(amidoamine) PAMAM dendrimers and zerovalent metals, such as gold and silver. Structure of {(Au(0))n−PAMAM} and {(Ag(0))n−PAMAM} gold and silver dendrimer nanocomposites was found to be the function of the dendrimer structure and surface groups as well as the formation mechanism and the chemistry involved. Three different types of single nanocomposite architectures have been identified, such as internal (‘I’), external (‘E’) and mixed (‘M’) type nanocomposites. Both the organic and inorganic phase could form nanosized pseudo-continuous phases while the other components are dispersed at the molecular or atomic level either in the interior or on the surface of the template/container. Single units of these nanocomposites may be used as building blocks in the synthesis of nanostructured materials.

Designing recombinant spider silk proteins to control assembly.

The consensus repeat sequence found in the dragline silk from the spider, Nephila clavipes, was redesigned to incorporate a redox trigger flanking the beta-sheet forming polyalanine sequences. The methionine redox trigger, in the oxidized state, was incorporated to prevent the formation of the beta sheets, while in the reduced state would not result in sterical limitations to beta sheet formation. A synthetic gene incorporating the trigger was constructed, cloned and then expressed in Escherichia coli. The purified protein, about 25 kDa, contained the expected amino acid composition and migration behavior on SDS-PAGE. The recombinant protein was analyzed by X-ray diffraction, TEM, electron diffraction and circular dichroism in both oxidized and reduced states. Based on the results, the incorporation of a redox trigger appears to be a powerful experimental strategy to explore the self-assembly of fibrous proteins such as silks.

Orientation of silk III at the air-water interface

A threefold helical crystal structure of Bombyx mori silk fibroin has been observed in films prepared from aqueous silk fibroin solutions using the Langmuir Blodgett (LB) technique. The films were studied using a combination of transmission electron microscopy and electron diffraction techniques. Films prepared at a surface pressure of 16.7 mN/m have a uniaxially oriented crystalline texture, with the helical axis oriented perpendicular to the plane of the LB film. Films obtained from the air-water interface without compression have a different orientation, with the helical axes lying roughly in the plane of the film. In both cases the d-spacings observed in electron diffraction are the same and match a threefold helical model crystal structure, silk III, described in previous publications. Differences in the relative intensities of the observed reflections in both types of oriented samples, as compared to unoriented samples, allows estimations of orientation distributions and the calculations of orientation parameters. The orientation of the fibroin chain axis in the plane of the interfacial film for uncompressed samples is consistent with the amphiphilic behavior previously postulated to drive the formation of the threefold helical silk III conformation.

Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin-organic solvent interfaces.

A banded morphology has been observed for Bombyx mori silk fibroin films obtained from an aqueous hexane interface; the period of the banding is approximately 1 microm. Morphology and diffraction from different regions of the banded structure suggest that it is a free surface formed by a cholesteric liquid crystal. Truncated hexagonal lamellar crystallites of B. mori silk fibroin have been observed in films formed in the surface excess layer of fibroin at the interface between aqueous fibroin and hexane or chloroform. Based on initial crystallographic evidence, a three-fold helical conformation has been ascribed to the fibroin chains within the crystals. The chain conformation and crystalline habit appear to be similar to the silk III structure previously observed at the air-water interface (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Valluzzi R, Gido S, Zhang W, Muller W, Kaplan D. Macromolecules 1996;29:8606-8614) but the crystalline packing is different. Diffraction data obtained for the crystallites are similar to diffraction behavior for a collagen-like model peptide. Diffraction patterns obtained from crystallized regions of the banded morphology can be indexed using the same unit cell as the hexagonal lamellar crystallites. Surfactancy of fibroin and subsequent aggregation and mesophase formation may help to explain the liquid crystallinity reported for silk, which is long suspected to play a role in the biological silk spinning process (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Willcox, P. J.; Gido, SP, Muller W, Kaplan DL. Macromolecules 1996:29:5106-5110; Magoshi J, Magoshi Y, Nakamura S. In: Kaplan D, Adams W, Farmer B, Viney C, editors, Mechanism of Fiber Formation of Silkworm. Washington, DC: American Chemical Society 1994:292-310; Magoshi J, Magoshi Y, Nakamura S. J Appl Polym Sci Appl Polym Symp 1985;41:187-204; Magoshi J, Magoshi Y, Nakamura S. Polym Commun 1985;26:309.).

Sequence-specific liquid crystallinity of collagen model peptides. I. Transmission electron microscopy studies of interfacial collagen gels.

The conformation, crystal structure and self-assembly behavior of three peptides with collagen-like repetitive sequences [(1) peptide GAPGPP: (Glu)(5)(Gly-Ala-Pro-Gly-Pro-Pro)(6)(Glu)(5); (2) peptide GVPGPP: (Glu)(5)(Gly-Val-Pro-Gly-Pro-Pro)(6)(Glu)(5); and (3) peptide GAPGPA: (Glu)(5)(Gly-Ala-Pro-Gly-Pro-Ala)(6)(Glu)(5)] were compared. The peptides were characterized using transmission electron microscopy, electron diffraction, environmental scanning electron microscopy, and Fourier transform ir spectroscopy in order to determine how the molecular geometry dictated by each sequence affects the spontaneous generation of long-range ordered structures. Samples of each peptide, at ambient temperature and at 5 degrees C, were examined as films dried from aqueous solution, air-water interfacial films, and chloroform-water interfacial films. Peptide GAPGPP prepared at 5 degrees C and dried from bulk solution was found to have a collagen-like triple-helical structure. A sinusoidally textured gel, suggestive of cholesteric behavior was observed for peptides GAPGPP and GVPGPP at the aqueous chloroform interface at 5 degrees C. Peptide GAPGPA also formed a gel, but less reproducibly and the sinusoidal texture was not as well defined. The periodicities of the sinusoidal textures were reproducibly 10 microm for peptide GAPGPP, 7 microm for peptide GVPGPP, and 6 microm for peptide GAPGPA. The differences in the periodicity of the banded structure and in the crystallization behavior of the three peptides is attributed to differences in the symmetry of the preferred packing arrangement for each peptide, as evidenced by electron diffraction from crystallites that coexist with the sinusoidal gel. These differences are believed to be a measure of the effective symmetry and shape of the molecular cross section.

Magnetically complexed collagen nanocomposites

Rare-earth ions with unfilled 4f shells, Gd3+ (J =  ) and Dy3+ (J =  ) are bound to glutamic acid residues at the ends of long linear oligopeptides derived from those in naturally occurring collagen proteins. The peptide, which folds into a triple helix about 10 nm long, consists of a sequence 36 amino acids long. The rare-earth–collagen complex self-assembles into layers through a smectic A liquid crystalline phase. The smectic layered structure is retained on drying, and the acidic peptide end blocks localize the rare-earth ions in sheets approximately 1–2 nm thick separated by the collagen-like peptide of the complex. X-ray crystallographic, scanning electron microscopy and optical studies demonstrate the liquid crystallinity of the peptides and the incorporation of the rare-earth ions into sheets between the smectic layers. Magnetic susceptibility (2 K ≤ T ≤ 300 K) and low-temperature isothermal magnetization (0 T ≤ H ≤ 5 T) show the rare-earth–collagen complexes are paramagnetic in the rare-earth concentration range studied (not more than 0.3–0.4 Gd or Dy ion per molecule). This technique of bonding rare-earth ions to oligopeptides, and using the oligopeptide mesophase behaviour to organize attached ions, can be used to tether other rare-earth and/or transition-metal ions, creating layered magnetic structures. The interlayer spacing is dictated solely by the intervening oligopeptide length.

Magnetically complexed tissue-mimicking peptides

We have synthesized oligopeptides based on insect silks with added metal ion complexing groups at the ends of the oligopeptides. These oligopeptides can be complexed with magnetic ions and used to chemically deliver them into a pattern of nanoscale magnetic ion-rich layers without thin-film deposition. Gd-, Dy-, and Cr-complexed oligopeptide materials were studied and were found to be well-behaved paramagnets. With higher concentrations of magnetic ions, magnetic coupling interactions may be possible.