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.

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.

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis.

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.

Wound Debridement by Continuous Streaming of Proteolytic Enzyme Solutions: Effects on Experimental Chronic Wound Model in Porcin.

Wound debridement for the removal of necrotic tissue is a crucial step in wound management. Enzymatic wound debridement is one example of a method currently used that removes necrotic tissue with proteases and offers selectivity without affecting healthy adjacent tissue. Proteolytic enzymes for wound debndement are commercially available as ointments. The authors previously proposed and demon- strated feasibility on small lab animals-an alternative mode of deliv- ery of proteolytic enzymes for wound debridement with continuous streaming of protease solutions. The present study describes the impact of streaming of papain solut ions, fort ified by the incorporation of hypertonic agents, onto an experimental larger chronic wound model in pigs. Debridement of approximately half of the necrotic tis- sue mass was achieved within 6 to 11 h of streaming of papain solu- tions onto these experimental wounds. No adverse effects or notice- able morphological changes to the wound surface or its immediate surroundings were noted, indicating enzyme selectivity and preference for attacking necrotic tissue. The mechanism of enzymatic attack on the necrotic tissue is also discussed. In the control group, streaming of the basic solution formula (devoid of papain) was performed-no debridement of necrotic tissue was noticed in this case. The results indicate that the streaming delivery mode for enzymatic debridement is a highly effective tool designed to be completed in a few sessions. thus paving the way for extension of its application in clinical trials on humans.

Protein-mediated nanoscale biotemplating.

Biomimetics--the concept of taking ideas from nature and implementing them in technology--has found particular use for the development of nanoscale materials. One such approach employs protein-mediated biotemplating for the nanostructuring of inorganic material. Recently, two key advances have been witnessed in this field. Firstly, the number of successfully employed biotemplates, including feasibility demonstrations of using three-dimensional crystalline structures, has been expanded. Secondly, the introduction of site-directed mutations on the protein template, or the display of peptides that exhibit effective biorecognition sequences for inorganic structures, has led to substantial improvements in our ability to control protein-mediated biotemplating. Taken together, these achievements will pave the way for the successful application of protein-mediated biotemplating in the future.

Monitoring the stability of crosslinked protein crystals biotemplates: a feasibility study.

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 molecules in the crystal is a complementary 3D array of voids made of interconnected cavities and exhibiting highly ordered porosity. The permeability of the porosity of chemically crosslinked enzyme protein crystals to low molecular weight solutes, was used for enzyme mediated organic synthesis and size exclusion chromatography. This permeability might be extended to explore new potential applications for protein crystals, for example, their use as bio-templates for the fabrication of novel, nano-structured composite materials. The quality of composites obtained from "filling" of the ordered voids in protein crystals and their potential applications will be strongly dependent upon an accurate preservation of the order in the original protein crystal 3D array during the "filling" process. Here we propose and demonstrate the feasibility of monitoring the changes in 3D order of the protein array by a step-by-step molecular level monitoring of a model system for hydrogel bio-templating by glutaraldehyde crosslinked lysozyme crystals. This monitoring is based on step-by-step comparative analysis of data obtained from (i) X-ray crystallography: resolution, unit cell dimensions and B-factor values and (ii) fluorescence decay kinetics of ultra-fast laser activated dye, impregnated within these crystals. Our results demonstrated feasibility of the proposed monitoring approach and confirmed that the stabilized protein crystal template retained its 3D structure throughout the process.

Screening of large protein libraries by the cell immobilized on adsorbed bead approach.

Screening of mutant libraries for in vitro enzyme evolution is carried out primarily by physical separation of the cells, followed by growth of individual clones and screening of biocatalytic activity on the basis of color or fluorescence signal development. Currently, most frequently employed methods are labor-intensive or require robotic equipment, resulting in screening limited to a relatively small fraction of the potential inherent in a given library. In this study we present a design, development, and feasibility demonstration of a new screening approach, providing convenient handling of large libraries consisting of 106 to 107 clones and screening based on a simultaneous enzymatic assay with commercially available substrates. This new screening method is based on the "cell immobilized on adsorbed bead" approach: the cell population to be screened is mixed with an excess of medium pre-equilibrated polyacrylamide beads, chemically derivatized to affect quantitative cell immobilization by adsorption. The resulting bead population, comprising of single cell on a bead or blank beads, is then immobilized on a solid glass support. After removal of the freely flowing liquid, the cells immobilized on the adsorbed beads are allowed to grow into microcolonies, utilizing the medium retained within the supporting hydrogel matrix. These colonies are subsequently equilibrated with chromogenic or fluorogenic substrate and screening is affected under a stereomicroscope, resulting in readily retrieved of the most active colonies. This technique may be particularly useful when the screened mutants are expressed and displayed on the cell surface, providing an active and homogeneous "naturally immobilized" enzyme population with minimal substrate diffusion limitations.