Structural Biology

The detailed knowledge about structure, interaction and organization of biological macromolecules, especially of proteins, is an essential requirement for understanding biological processes and thus for the development of biotechnological products. Only a deep structural insight into the atomic world of biological important molecules allows the rationally guided design of pharmaceuticals, diagnostics, enzymes and production systems.

Therefor one of the major focuses of our group is the structural biology in different areas of research, including the following technologies and approaches:

 

  • Protein crystallization and rational crystal analysis
  • X-ray structure analysis
  • Analysis of protein/ligand-complexes

 

Active sites of betalactamases in complex with an inhibitor and /or with zinc ions; cartoon representation of an industrially relevant P450 monooxygenase for steroid modification

 

Protein crystallization

 

Protein crystallization is the major bottleneck within the complete process of X-ray structure determination. Often numerous screening experiments are required to obtain suitable crystals for X-ray diffraction analysis. During the last years, several large structural genomics initiatives have put great effort into this field of research, focusing mainly on high-throughput screening technologies, but still protein crystallization remains a difficult task and new approaches are required, to solve this problem and to open the door to the atomic level world of protein structures completely.

 Therefor our group develops new methods for a rationally guided protein crystallization, based on physical measurements  as well as on computational predictions. This includes for example light scattering approaches and fluorimetry techniques for the determination of protein-protein and protein-ligand interaction and stability parameters under different physicochemical conditions. Computational predictions are usually based on data mining results from a variety of experiments. New technologies are furthermore optimized and implemented into high-throughput devices to build up a rationalized and automated protein crystallization suite and to offer a highly efficient and reproducible structure determination service to customers

 

Protein crystals of different shape and quality 

 

Birefringence analysis of crystallization screens

 

The most important optical analysis technique used to evaluate the crystallization pipeline outcomes is the quantitative polarization microscopy. Based on multiple pictures taken under different polarization angles of incident light, optical anisotropies within each droplet can be quantitatively determined via birefringence. While this technique is applicable in many different areas, in protein crystallization it is mostly used to answer two questions. Because crystals are optically anisotropic bodies, birefringence analysis can help to distinguish between crystalline and amorphous phases, which is often problematic in the case of microcrystalline precipitates or sea-urchins. Furthermore it is used to evaluate the quality of protein crystals prior to x-ray diffraction, where high-quality monocrystals are required. The use of a quantitative polarization microscope (Taorad), which is optimized for the automated analysis (droplet detection, automated picture taking and analysis) of our crystallization plates, allows an effective and minimally invasive evaluation of crystallization screening outcomes and thus enables a fast and successful protein crystallization for x-ray structure determination and other applications (e.g. formulation of pharmaceutical proteins).

 

Polarization Microscope MP1 (Taorad); protein crystals of different quality with corresponding diffraction pattern; discrimination between microcrystals, sea-urchins, precipitates and phase separations using quantitative polarization microscopy

 

X-ray diffraction, structure solution and analysis

 

Until now, the most successful method for the determination of macromolecular structures on an atomic level is the x-ray crystallography. Our in-house x-ray diffraction unit (wavelength of 1.54 A) in combination with the powerful crystallization pipeline allows us to solve structures of biological macromolecules, especially proteins and their complexes with small-molecule ligands or other proteins.

But structure analysis means not only structure solution by x-ray crystallography. Based on the solved structures we use molecular dynamics simulation to find potential ligands for or interactions with a target protein (e.g. beta-lactamase inhibitors). Our structural biology pipeline then gives us the opportunity to validate the computational findings experimentally using x-ray crystallography as well as other biophysical analysis approaches.

 

X-ray diffraction unit, protein crystal diffraction pattern, model building in coot

 

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