The Contribution of Polystyrene Nanospheres towards the Crystallization of Proteins

Abstract

BackgroundProtein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins.MethodologyThe effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT), and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times.ConclusionsAll in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.

Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase fromHalorhodospira halochoris

“…The best protein concentration for crystallization experiments was approximately 9 mg ml À1 . Polystyrene nanoparticles (Kallio et al, 2009) were tested in some crystallization experiments as additives in the crystallization droplet. Nanospheres had a positive effect on crystal growth and we were able to grow large crystals in less time (1-7 d).…”

Protein Crystallization by Anodic Porous Alumina (APA) Template: The Example of Hen Egg White Lysozyme (HEWL)

“…In order to control heterogeneous nucleation of lysozyme crystals, hair cuticles [8] or ad hoc engineered structured surfaces such as Poly-L-Lysine modified glass substrate [9], chemically modified mica surfaces [10] or other chemically modified patterns [11], xanthenes dyes [12], polystyrene nanospheres [13], porous glass [14], porous silicon [15] and fluorinated layered silicate (which is a phyllosilicate with a parallel two-dimensional lamellar structure; Ino et al, 2011) [16] can be exploited. In the last work, the fluorine atoms were considered responsible for driving the nucleation process.…”

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

“…Favourable crystallization conditions were found using the modified PEG3350 series for antibodies (Valjakka et al, 2000). 5F2 Fab crystals suitable for X-ray analysis were obtained by combining the streak-seeding method with nanospheres (Kallio et al, 2009). Briefly, the crystallization droplets contained 2 ml of protein solution (8.3 mg/ml in 20 mM HEPES buffer, pH 7.0), 0.5 ml of testosterone solution (5 mM in 50% ethanol), 1 ml of nanosphere solution (50 nm in diameter, 1:1 dilution to water) and 2 ml of precipitant solution (12% v/w PEG3350, 0.1 M sodium citrate at pH 4.7).…”