Investigating the Effects of Nanogels in Promoting Protein Crystallization.

X-ray crystallography is still the most widely used and versatile method for structural studies of biological macromolecules. This study concerns the application of nanogels to facilitate protein crystallization, a prerequisite for X-ray crystallography. Nanogels (NGs) are nano-sized, highly crosslinked polymeric particles that have been extensively studied for chemical catalysis and drug delivery but not for protein crystal nucleation. The efficacy of six types of nanogels (three N-isopropylacrylamide-based and three acrylamide-based) was tested, with promising results. They were subsequently functionalised with active hydroxyl groups for further testing. Both functionalised and non-functionalised nanogels were tested on model (trypsin, thaumatin, proteinase K, ferritin and catalase) and target proteins (glulisine, α-crustacyanin and acriflavine resistance protein subunit AcrB) using both manual and automated techniques. All nanogels were found to be effective in promoting protein crystallization in both screening and optimization trials, giving crystal 'hits' that would have otherwise been missed. Overall, the functionalised nanogels were more effective. Nanogel effects are proposed to be due to a combination of surface porosity and surface chemistry.