Polymers as Heterogeneous Growth Promoters for Protein Crystallization.

Abstract

Obtaining suitable single crystals for X-ray diffraction remains a major bottleneck in the structural characterization of new compounds. Nowhere is this more apparent than in structural biology where the challenges of crystal growth are considerable. In cases where traditional growth methods fail to yield suitable protein crystals, the target is often reconsidered, rather than the crystallization approach. In the studies described in this thesis, polymer-induced heteronucleation (PIHn), a powerful technique well-established in the realm of small molecule crystallization, is tailored to meet the challenges inherent to protein crystallization. Based on the premise that polymers designed for small molecule crystallization may not be best suited to facilitating biomolecule crystallization in PIHn, the heteronucleant composition in PIHn was modified by introducing less hydrophobic crosslinking agents, which led to increased crystal size and new form access. The polymers were additionally redeployed in a variety of formats suitable for sitting and hanging-drop vapor diffusion crystallizations. The power and utility of these advances was demonstrated through the application of PIHn to bovine liver catalase (BLC) and concanavalin A (conA). For BLC, this approach led to increased crystal size and ultimately the first X-ray crystal structure of a crystal form of BLC that was previously too small for structural characterization. Additionally, in the presence of the heteronucleants two novel forms of conA were discovered and analyzed using single crystal X-ray diffraction. Forming single crystals does not ensure structural characterization. Crystal harvesting often leads to crystal damage for delicate protein crystals, which can be amplified when using PIHn due to the propensity for crystals to adhere to the heteronucleants. This led to a growth strategy to allow nucleation on crystal mounts coated with polymer heteronuclei. In order to achieve selective control of nucleation, the surface chemistry of the crystal mount was modified using different heteronuclei. This method successfully controlled crystal growth for conA and BLC. A conA crystal grown directly on the crystal mount was characterized using X-ray diffraction, illustrating that the quality of the crystal was not negatively impacted by the presence of the functionalized crystal mount.