Polyoxometalate facilitated protein crystallization

Polyoxometalates (POMs) represent a large class of discrete polynuclear metal-oxo anions used for many different applications in catalysis, as sensors, in bio- and nanotechnology and in medical and material sciences. Yet there are very few studies that explore the potential of POMs to facilitate protein crystallization by looking specifically into the interaction between different types of POMs with proteins. This grant project is designed to make a contribution towards the fundamental molecular understanding on the interaction of POMs with proteins. Structurally knownPOMs (Na6[V10O28] 18H2O,Na6[TeW 6O24] 22H2O, Na14[NaP5W 30O110] 31H2O and Na6[P2W 18O62] 18H2O) and new POMs (Anderson-, Keggin-, and Lindqvist-based POMs) as well as hybrid POMs will be synthesized and characterized by different spectroscopic techniques and X-ray structure analysis with the aim to investigate the water solubility and the range of pH-stability. The elucidation of the crystal structures of several differing POM-protein complexes is the main goal. For the analysis of the structures a combination of protein purification, biochemical characterization, molecular biology and X-ray structure determination are the methods of choice. The proteins of interest are hen-egg white lysozyme, proteinase K, humanapo-transferrin, humanserumalbumin,myosin, polyphenol oxidase, hemocyanin, and the membrane protein bacteriorhodopsin. The outcome of this project will give novel insights to fundamental physical properties of polyoxometalates as well as insights into POM-protein interactions which is lacking in respective research community. With this highly interdisciplinary project a commercially available POM based crystallization screening set is intended to be developed. 1

Protein crystallography is still the most widely used method to obtain structural information on target proteins and protein-target interactions within the atomic resolution range. These structural information are indispensable for the understanding of the majority of biological processes and thus also of drug-target interactions. However, obtaining crystals that diffract to high resolutions represents the most essential but also the narrowest bottleneck in X-ray crystallography as this process is basically very time- and resource-consuming with no guarantee of success. The use of additives is an established process to facilitate or even enable the crystallization of proteins, however, their use is largely restricted to certain proteins or conditions. Therefore, there is a quest for a more universal additive addressing a wider range of proteins which is able to crystallize yet structurally unknown proteins. Therefore, we tested several compounds that belong to the class of polyoxometalates (POMs) for their suitability as crystallization agent. POMs are described as clusters of transition metal ions (molybdenum, tungsten, vanadium and niobium) and oxygen ions exhibiting an overwhelming diversity in size and structure with outstanding properties and functions. Our investigations revealed that the Anderson-Evans polyoxotungstate [TeW 6O24]6- (TEW) was the most suitable POM archetype in this regard as it led to the crystallization of two previously unknown proteins, polyphenol oxidase from mushroom (abPPO4) and aurone synthase from Coreopsis grandiflora (cgAUS1), and to the crystallization of the model protein hen-egg white lysozyme into a new crystal form and to a new crystal packing in the crystal structure of the HSP70 nucleotide binding domain. Due to these promising results and its unique properties, TEW was identified as a powerful crystallization additive exhibiting major advantages over commonly used additives. Due to its six-fold negative charge, TEW binds to positively charged protein patches but also to polar and uncharged regions, thus, addressing in principle all proteins possessing such sites. In this way TEW acts as a kind of inorganic glue as it, among others, connects equally (positively) charged proteins facilitating crystal lattice formation. TEW is highly stable under most crystallization conditions and it exhibits a huge entropic contribution to the crystallization energy by releasing large amounts of water molecules during crystal contact formation and thus energetically favors the crystallization event. Furthermore, by being a tungsten metal cluster it provides valuable anomalous signals, which are necessary for the phasing of proteins that lack suitable homologues for molecular replacement. All these features and results demonstrate that TEW, in many respects, is predestined as a powerful crystallization additive and crystallographers will benefit from its usage. Therefore, TEW was successfully established on the market and is now available as part of a crystallization screen (Jena Biosciences) and thus commercially accessible for all crystallographers.