Manufacture of stable palladium and gold nanoparticles on native and genetically engineered flagella scaffolds

The use of bacterial flagella as templates for the immobilization of Pd and Au nanoparticles is described. Complete coverage of D. desulfuricans flagellar filaments by Pd(0) nanoparticles was obtained via the H(2)-mediated reduction of Pd(NH3)4]Cl2 but similar results were not obtained using HAuCl4. The introduction of additional cysteine-derived thiol residues in the E. coli FliC protein increased Au(III) sorption and reduction onto the surface of the flagellar filament and resulted in the production of stabilized Au(0) nanoparticles of approximately 20-50 nm diameter. We demonstrate the application of molecular engineering techniques to manufacture biologically passivated Au(0) nanoparticles of a size suitable for catalytic applications.     

Re‐structuring protein crystals porosity for biotemplating by chemical modification of lysine residues

Protein crystals are routinely prepared for the elucidation of protein structure by X‐ray crystallography. These crystals present an highly accurate periodical array of protein molecules with accompanying highly ordered porosity made of interconnected voids. The permeability of the porous protein crystals to a wide range of solutes has recently triggered attempts to explore their potential application as biotemplates by a controlled “filling” process for the fabrication of novel, nano‐structured composite materials. Gaining control of the porosity of a given protein crystal may lead to the preparation of a series of “biotemplates” enabling different ‘filler’/protein content ratios, resulting in different nanostructured composites. One way to gain such control is to produce a series of polymorphic forms of a given “parent‐protein” crystal. As protein packing throughout crystallization is primarily dominated by the chemical composition of the surface of protein molecules and its impact on protein–protein interactions, modification of residues exposed on the surface will affect protein packing, leading to modified porosity. Here we propose to provide influence on the porosity of protein crystals for biotemplating by pre‐crystallization chemical modification of lysine residues exposed on protein's surface. The feasibility of this approach was demonstrated by the serial application of chemical “modifiers” leading to protein derivatives exhibiting altered porosity by affecting protein “packing” throughout protein crystallization. Screening of a series of modifying agents for lysine modification of hen egg white lysozyme revealed that pre‐crystallization modification preserving their positive charge did not affect crystal porosity, while modification resulting in their conversion to negatively charged groups induced dramatic change in protein crystal's packing and porosity. Furthermore, we demonstrate that chemical modification of lysine residues affecting modified protein packing may be simultaneously performed with the crystallization process: aldehydes generating Schiff base formation with protein's lysine residues readily affected modified protein packing, resulting in altered porosity. Our results demonstrate the feasibility of the use of site directed chemical modifications for the generation of a series of protein crystal exhibiting different porosities for biotemplating, all derived from one “parent” protein. Biotechnol. Bioeng. 2011; 108:1–11. © 2010 Wiley Periodicals, Inc.     

Rational shape engineering of the filamentous protein γ prefoldin through incremental gene truncation

An enticing possibility in nanotechnology is to use proteins as templates for the positioning of molecules in regular patterns with nanometer precision over large surface areas. However, the ability to redesign protein quaternary structure to construct new shapes remains underdeveloped. In the present work, we have engineered the dimensions of a filamentous protein, the γ prefoldin (γ PFD) from the hyperthermophile Methanocaldococcus jannaschii, and have achieved controllable attachment of filaments in a specific orientation on a carbon surface. Four different constructs of γ PFD were generated in which the coiled coils extending from the association domain are progressively truncated. Three of the truncation constructs form well‐defined filaments with predictable dimensions according to transmission electron microscopy. Two of these constructs had 2D persistence lengths similar to that of γ PFD at 300–740 nm. In contrast, the 2D persistence length of the shortest truncation mutant was 3500 nm, indicating that the filament adsorbs along a different axis than the other constructs with its two rows of coiled coils facing out from the surface. The elastic moduli of the filaments range from 0.7–2.1 GPa, similar to rigid plastics and within the lower limit for proteins whose primary intermolecular interaction is hydrogen bonding. These results demonstrate a versatile approach for controlling the overall dimensions and surface orientation of protein filaments, and expand the toolbox by which to tune two overall dimensions in protein space for the creation of templated materials over a wide variety of conditions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 496–503, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Modification of protein crystal packing by systematic mutations of surface residues: Implications on biotemplating and crystal porosity

Bioinspired nano‐scale biotemplating for the development of novel composite materials has recently culminated in several demonstrations of nano‐structured hybrid materials. Protein crystals, routinely prepared for the elucidation of protein 3D structures by X‐ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein “building blocks” in the crystal, a complementary 3D array of interconnected cavities—voids array, exhibiting highly ordered porosity is formed. The porous arrays of protein crystal may serve as a nano‐structured, accurate biotemplate by a “filling” process. These cavities arrays are shaped by the mode of protein packing throughout the crystallization process. Here we propose and demonstrate feasibility of targeting site specific mutations to modify protein's surface to affect protein crystal packing, enabling the generation of a series of protein crystal “biotemplates” all originating from same parent protein. The selection of these modification sites was based on in silico analysis of protein–protein interface contact areas in the parent crystal. The model protein selected for this study was the N‐terminal type II cohesin from the cellulosomal scaffold in ScaB subunit of Acetivibrio cellulolyticus and mutations were focused on lysine residues involved in protein packing as prime target. The impact of systematically mutating these lysine residues on protein packing and its resulting interconnected cavities array were found to be most significant when surface lysine residues were substituted to tryptophan residues. Our results demonstrate the feasibility of using pre‐designed site directed mutations for the generation of a series of protein crystal biotemplates from a “parent” protein. Biotechnol. Bioeng. 2009; 104: 444–457 © 2009 Wiley Periodicals, Inc.