Abstract
The limiting factor in protein crystallography is still the production of high-quality crystals. In this regard, the authors have recently introduced hexatungstotellurate(VI) (TEW) as a new crystallization additive, which proved to be successful within the liquid-liquid phase separation (LLPS) zone. Presented here are comparative crystal structure analyses revealing that protein-TEW binding not only induces and stabilizes crystal contacts, but also exhibits a significant impact on the solvent-driven crystallization entropy, which is the driving force for the crystallization process. Upon the formation of TEW-mediated protein-protein contacts, the release of water molecules from the hydration shells of both molecules, i.e. TEW and the protein, causes a reduced solvent-accessible surface area, leading to a significant gain in solvent entropy. Based on the crystal structures of aurone synthase (in the presence and absence of TEW), insights have also been provided into the formation of a metastable LLPS, which is caused by the formation of protein clusters, representing an ideal starting point in protein crystallization. The results strongly encourage the classification of TEW as a valuable crystallization additive.
Highlights
X-ray crystallography is currently the primary method for the structure determination of proteins, which is reflected by the content of the Protein Data Bank (PDB, http:// www.rcsb.org), with $90% of its entries resulting from this method
We describe the contribution of TEW to the crystallization entropy via determination of the solventaccessible surface area (ASA) and compare all three protein– TEW structures to demonstrate the particular aptitude of TEW as a powerful crystallization tool
Cryst1 and CrystTEW crystallized in the space group P1211, whereas Cryst2 was obtained in the space group P1, containing different numbers of protein monomers per asymmetric unit (ASU), viz. Cryst1 had four monomers, Cryst2 had eight monomers, and CrystTEW had two monomers
Summary
X-ray crystallography is currently the primary method for the structure determination of proteins, which is reflected by the content of the Protein Data Bank (PDB, http:// www.rcsb.org), with $90% of its entries resulting from this method. It has been demonstrated that short-range forces between protein molecules and the formation of protein clusters can result in an LLPS (Stradner et al, 2004). Experiments revealed that the critical temperature for LLPS formation is drastically lowered by the addition of Y3+ and the results implied that the cation-induced LLPS is an entropy-driven process owing to the release of hydration solvent molecules (Matsarskaia et al, 2016). The protein crystals of the three proteins that were cocrystallized with TEW were obtained either within (cgAUS1 and HEWL) or very close to (abPPO4) the LLPS zone. A similar behavior of TEW was observed during cocrystallization with HEWL, resulting in a new crystal form and confirming the suitability of TEW as a crystallization additive (Bijelic et al, 2015). We describe the contribution of TEW to the crystallization entropy via determination of the solventaccessible surface area (ASA) and compare all three protein– TEW structures to demonstrate the particular aptitude of TEW as a powerful crystallization tool
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