Expertise in Structural Biology
X-ray crystallography is a powerful technique to unravel the three-dimensional architecture of large macromolecules such as proteins or nucleic acids but also of multi-component macromolecular complexes such as the ribosome. The method utilizes the diffraction of X-rays by crystals which represent a regular arrangement of the macromolecule or macromolecular complex in a periodic lattice to give precise data about the relative position of each atom of the molecules in the crystal. The most important and often rate-limiting step in X-ray crystallography is the crystallization of the macromolecule of interest. Exposure of the macromolecular crystal to X-ray radiation produces a diffraction pattern that can be translated into the arrangement of atoms in the molecule by a mathematical process thereby unraveling the detailed architecture of the molecule. This detailed structural information can be used to unravel the catalytic mechanism of a protein, to identify the binding sites for a certain substrate or inhibitor or even to recognize the molecular contacts in protein-protein or protein-nucleic acid complexes.
Principle of structure determination by X-ray crystallography. First step is the crystallization of the protein. When the crystals are exposed to X-ray radiation a typical diffraction pattern resulting from the scattering of X‑rays on the planes of the crystal lattice is obtained. From this pattern electron density maps are computed. Finally, a model of the protein is constructed from these electron density maps.
Fluorescence polarization: Most catalytic or regulatory pathways operate as networks consisting of permanent or temporary complexes. Quantitative analysis of protein-protein interactions in these complexes unraveling association kinetics and stability of the complex can be obtained from fluorescence polarization measurements. In these studies one of the reaction partner is labeled with a rigid fluorophore e.g. by fusion of the protein of interest to a fluorescent protein (GFP) derived from the marine jellyfish Aequorea victoria. When a cuvette containing the GFP fusion protein is exposed to linear polarized light only those molecules are excited which have their absorption dipole aligned with the direction of the electric vector of the incident light. In a homogenous aqueous solution the direction of the polarized light that is emitted by the fluorophore differs from the incident beam direction due to the rotational diffusion of the excited molecules in the medium. An increase in the molecular weight of the fluorescent molecule e.g. due to ligand binding, reduces rotational diffusion and is detected by an increase in fluorescence polarization which can be used to assess complex formation and stability.
Monitoring complex formation by fluorescence polarization. (A) Excitation of a fluorescent molecule by linear polarized light. During the life time of the excited state the fluorescent molecule rotates resulting in a shift in the direction of the emitted polarized light compared to the incident beam direction. (B) Formation of a complex with the fluorescent protein decreases the rotation of the fluorophore which is detected by an increase in fluorescence polarization.