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

Abstract

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.

Protein crystal based materials for nanoscale applications in medicine and biotechnology

“…Accordingly, there have been multiple attempts to modify protein scaffolds to optimize specific properties related to transport, catalysis, and templating. In 2009 and 2011, Felix Frolow and Amihay Freeman's group used both systematic mutation (Wine, Cohen-Hadar, Lamed, Freeman, & Frolow, 2009) and chemical modification (Cohen-Hadar, Lagziel-Simis, Wine, Frolow, & Freeman, 2011) of pore surface residues to alter the porosity of HEWL crystals in the interest of biotemplating applications. Furthermore, they determined that simple addition of various metal ions (Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ ) to the crystal growth environment could provide a strategy to "fine tune" the porosity of the resulting crystal (Wine et al, 2010).…”

Protein-Mediated Biotemplating on the Nanoscale

“…An alternative route to affect parent protein–protein molecular interactions affecting crystal “packing” was demonstrated by Cohen-Hadar et al [84], using the chemical modifications of the lysine amino groups of the parent protein, as shown in Figure 6 for lysozyme crystals.…”

Modification of Amino Groups

“…Modifies amino groups to a strongly basic form; may be used as a method to survey the exposure of amino groups in protein complexes (Liu and Reilly, 2009;Cohen-Hadar et al, 2010) Phenylthiocarbamylation (Mindt et al, 2008;da Silva Freitas et al, 2013) BirA-catalyzed biotinylation Biotin + ATP in presence of BirA Biotin + ATP + protein with tag sequence GLNDIFEAQKIEWHE BirA forms biotinyl-5 -AMP in its own active center, then transfers biotin to ɛ-amino group of Lys with specificity favoring a particular peptide "tag" (Beckett et al, 1999) heterogeneity caused by modification of a fraction of the molecules complicates downstream characterization of a bioconjugate by splitting the mass of its base protein into two or more values.…”