Membrane protein structural characterization

    Once conditions have been identified to produce, purify and stabilize the target membrane protein, its structural characterization can be initiated. Due to the size of our target membrane proteins, X-ray crystallography is currently the most suitable approach to obtain high-resolution structures. In addition, this is a well-accessible technique. X-ray crystallography requires the availability of crystals that are well-ordered. Due to their limited hydrophilic surfaces, which are important for forming contacts within a crystal lattice, membrane proteins are often difficult to crystallize and improving the diffraction quality of a membrane protein crystal can be troublesome.
    We counter low crystallizability and poor ordering by using crystallization chaperones. Crystallization chaperones are stable, soluble proteins that are engineered or selected to bind specifically to a target protein. Crystallization chaperones increase the chances of obtaining well-diffracting crystals. Their binding to the target protein increases the hydrophilic surface available for formation of crystal contacts. Furthermore, they can reduce the conformational heterogeneity of the target protein and stabilize it in a certain conformation.

    Several different types of crystallization chaperones are available nowadays, ranging from animal-derived Fab fragments up to engineered synthetic binding scaffolds such as DARPins, anticalins, monobodies and fynomers. We have succesfully used crystallization chaperones based on camelid single-chain antibodies, also known as Nanobodies or VHH’s, in collaboration with the Steyaert lab in Brussels, Belgium. A VHH consists of the variable domain of a heavy chain-only antibody. These single-chain antibodies are unique and only found in camelids and cartilaginous fish such as sharks. Nanobodies are specifically suited for intimately interacting with membrane proteins as they contain a relatively long complementarity determining region (CDR3) that can penetrate into cavities such as membrane-embedded substrate binding sites. The Nanobody-technology combines the thus far unrivaled potential of an immune system to generate affinity-matured, highly specific antibodies with in vitro selection strategies and bacterial production. This results in binders of high quality that can easily be produced in large amounts.

    In addition to this, we are collaborating with the Seeger lab at the Institute of Medical Microbiology and Dr. Saša Štefanić at the Institute of Parasitology, in Zurich, Switzerland, to develop accelerated procedures for obtaining crystallization chaperones.