Novel vanadates as additives in protein crystallization

APPLICANT: RNDR. LUKŠ KRIVOSUDSK, PHD. CO-APPLICANT: UNIV.-PROF. DR. ANNETTE ROMPEL Polyoxometalates are transition-metal clusters exhibiting a wide range of structural diversity and applications, such as in catalysis, magnetism, medicine, electrochemistry, luminescence and material science. However, in the last two decades it has been shown that the interaction of polyoxometalates with proteins may be exploited for utilizing them as an important tool for protein crystallization and subsequent crystal structure elucidation. One of the most prominent employment of POMs in protein crystallography was their use during early stages of the structure elucidation of the ribosome, a project which was later awarded the Nobel Prize in chemistry in 2009. The knowledge of protein structures is of vital importance for understanding their functions in organisms, designing drugs (or drug discovery) and for other various applications in medicine, biochemistry and technology. The here presented project aims to synthesize novel polyoxometalates, mostly vanadates and vanadium-containing molybdates. These polyoxometalates will be functionalized by introducing different heteroatoms into the structure (phosphorus, sulfur, fluorine) and/or by attaching organic functional groups to expand the options for possible interactions with proteins. Our main goal is to prepare by developing new synthetic methods and structurally characterize new polyoxovanadates with the potential to function as crystallization additives, especially for the crystallization of membrane proteins. Membrane proteins are of important interest, since 1/3 of all human proteins are membrane proteins and therefore they contain the large majority of drug targets. However, the crystallization of this protein family is very difficult because of their membrane associated hydrophobic regions, which is the reason why only 2% of all solved protein structures are membrane proteins. Thus, the results of this project will not only enrich the polyoxometalate chemistry but also increase the knowledge about (membrane) proteinpolyoxometalate interactions, which will hopefully lead to a polyoxometalate-based strategy for membrane protein crystallization. The polyoxometalate- protein interactions will be examined by the means of X-ray diffraction analysis as well as studied in solution by various methods available at the host institute.

The project was aimed at the synthesis and structural characterization of new polyoxometalates that can be used as additives in protein crystallography or possess biological activity. Polyoxometalates are discrete polynuclear metal-oxo anions with a wide variety of structures and distinct chemical and physical properties. A series of structurally different types of compounds involving vanadium (vanadates) was prepared. A new mixed vanadium-molybdenum anion whose existence was proposed twenty years ago by nuclear magnetic resonance spectroscopy was prepared using a unique strategy based on vanadium peroxido complexes. The method represents an easy and environmentally friendly route to vanadates because it employs aqueous solutions only. The initial peroxido complexes of vanadium release only oxygen as a side product. Two decavanadates functionalized by inorganic biogenic cobalt and copper cations were prepared and studied for their interaction with different proteins, such as human serum proteins, model proteins used for protein crystallography or a sweet protein thaumatin. The spectroscopic investigation revealed that the modified decavanadate interacts differently with the proteins and the pilot investigations will be further developed to find optimal conditions for the use of this type of compounds as promoters or enhancers of protein crystallization. The knowledge of protein structures is of vital importance for understanding their functions in organisms, designing drugs (or drug discovery) and for other various applications in medicine, biochemistry and technology. It was found that inorganic functionalization of polyoxometalates by attachment of biogenic metals such as cobalt and copper may tune their interaction with the proteins making them suitable candidates for crystallization additives for a very differentiated variety of proteins. In a collaboration with hosting Professor Manuel Aureliano (University of Algarve, Portugal) different polyoxotungstates were studied as inhibitors of ATPase - an enzyme producing energy in organisms. Similar study was performed also using a robust phosphotetradecavanadate anion that showed the highest activity compared to other vanadates. The results reveal that some of the studied compounds are potent inhibitors of the enzymes even under almost physiological conditions and therefore should be further studied as drugs targeting this enzyme. Several compounds were also studied for their antimicrobial activity. The results of the project were presented at the International Vanadium Symposium 2018 held in Montevideo, Uruguay, which is a biannual meeting collecting the most recent and outstanding achievements in vanadium chemistry. The concept of the use of polyoxometalates as protein additives was introduced also in the form of an invited lecture given at the biannual conference Advances in Inorganic Chemistry 2018 held in Levoca, Slovakia.