Polyoxoions for protein crystallization

Applicant: Dr. Nadiia Gumerova (Research fellow, Department of Inorganic Chemistry, Donetsk National University, Ukraine) Co-applicant: Prof. Annette Rompel (Head of the Department of Biophysical Chemistry, University of Vienna, Austria) Protein crystallography represents at present the most productive and most widely used method to obtain structural information on target proteins and protein-ligand complexes at the atomic resolution range. This knowledge is essential for understanding the biology, chemistry and biochemistry of protein function. Here we address the at present unpredictability of the crystallization of proteins, which represent the essential step to perform any structural study by protein crystallography and still depends basically on trial and error, can take up to several years and requires typically milligram amounts of highly pure and homogenous protein preparations. Polyoxometalates (POMs), discrete polynuclear metal-oxo anions with a fascinating variety of structures and unique chemical and physical properties, were repeatedly reported to positively affect the crystallization behavior of proteins and act as a valuable tool in crystallization and crystallographic studies via derivatization. The general aim of the proposed project is the synthesis and characterization of inorganic and hybrid organic-inorganic polynuclear metal-oxo ions (cations and anions) to investigate their potential application in the field of protein crystallography. The innovativeness and unique character of the project lies in the usage of both anionic and cationic POMs for co-crystallization with proteins. The positive effect that anionic polyoxometalates have shown so far urges the need for applying cationic POMs in protein crystallization. The combination of organic fragments, which may adopt different conformation, and inorganic polyanions with fairly rigid cagelike structures will improve the interaction between the macromolecule which is very often the basis for successful crystal formation. The proposed study has a broad intersectoral character from inorganic chemistry via organic derivatization to biochemistry applying biophysical characterization methods and will pose if successful - a breakthrough to future studies in the field of integrated macromolecular crystallography. The applicant (Dr. Nadiia Gumerova) studied special aspects of Anderson-type structure (Ph.D. at Donetsk National University, Ukraine) and hence has expert knowledge on both polyoxometalate synthesis and their solution behavior. The host (Prof. Annett Rompel) is a leading researcher in the metalloprotein crystallography. The combined expertise and ideal experimental facilities available at the University of Vienna gives the project the very best chance of success.

Polyoxometalates (POMs) are discrete polynuclear metal-oxo anions with a fascinating variety of structures and unique chemical and physical properties. Their full potential can be optimized by enhancing their biocompatibility through organic functionalization with bioactive moieties. In recent years, POMs emerged as a promising group of protein crystallization additives. Protein crystallography is the most widely used method for determining the molecular structure of proteins and obtaining structural information on proteinligand complexes at the atomic level. Hybrid organic-inorganic polyoxotungstates (POTs) address different kinds of interactions between a POM and a biomolecule. The possibility to decorate inorganic POMs, in particular the Anderson type, with organic moieties opens the door for the synthesis of specifically tailored POMs. During the project, Cr-centered Anderson POTs were organically functionalized for the first time from two sides and showed that the double-side functionalization is possible even in the case when the parent inorganic anion state exhibits only three protons in the solid. It was generally accepted that it is not possible to graft triol ligands onto the Anderson anions, which represent unprotonated 3-O atoms in their structures as confirmed by single crystal X-ray analysis in the solid state. The new compound can be seen as a game changer for alkoxylation of A-type Anderson POTs exhibiting new properties that differ from those of the already existing hybrids based on the protonated Anderson archetype. The way to overcome the resistance of the A-type towards functionalization is the use of a temperature controlled hydrothermal synthetic route, which has not been applied before in the tris-functionalization of the Anderson archetype. Applying the proposed method, mixed-type Anderson POMs with a protonated and proton-free side can be asymmetrically functionalized in two steps, in which two different organic groups are selectively grafted onto each side of the cluster. Since the introduced organic ligands present active functional groups, Anderson-type hybrids can be utilized as precursors for further post- functionalization via imine, amide, or ester bond formation for further use in protein crystallography. Complementary to the design of POMs for protein crystallization the project interests were extended on other highly important biological activities such as antibacterial activity and inhibition of P-type ATPase and mushroom tyrosinase.