Monitoring the stability of crosslinked protein crystals biotemplates: A feasibility study

Abstract

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.

Purification, crystallization and preliminary X-ray crystallographic studies of the complex between Smc5 and the SUMO E3 ligase Mms21

“…2a) and the SeMet crystal diffracted to 7 Å resolution. To improve the diffraction, 5 ml of the cross-linking reagent glutaraldehyde was added to the 500 ml reservoir solution (Cohen-Hadar et al, 2006). Crystals were equilibrated at 293 K for 5 min, 10 min, 30 min, 2 h, 3 h and 4 h before being mounted on the loop.…”

Protein crystal based materials for nanoscale applications in medicine and biotechnology

“…Soon after, they demonstrated the enzymatic activity of GA cross‐linked carboxypeptidase‐A crystals in aqueous solutions different from that of the crystallization environment (Quiocho & Richards, ). Since then, aldehydes (Figure a), specifically GA, have been the dominant cross‐linking agents for the stabilization of protein crystals (Cohen‐Hadar et al, ; N. K. Khalaf et al, ; Yan et al, ). Despite the ubiquity of GA cross‐linking, the chemical basis is incompletely understood.…”

Cross-linked protein crystals by glutaraldehyde and their applications

“…Protein crystals possess a three-dimensional ordered arrangement of protein molecules. 93 Cohen et al 94 dissolved CLPCs in aqueous ethylene glycol, induced polymerisation by the addition of ammonium persulfate, and then retrieved the crystals. Cvetkovic et al 91 investigated the transport of uorescein in lysozyme crystals by confocal laser scanning microscopy and found that the diffusion rate was highly related to the crystal structure.…”

“…Currently, the CLPCs have attracted a great deal of research interest as biological templates for the preparation of novel nano-structured composite materials. 94 Since then, the feasibility of using CLPCs as effective bio-templates for the construction of novel patterned composite materials has attracted pronounced attention.…”