Contact

Dr. Ronald Frank

phone +49 (0)30 9406 3066
fax +49 (0)30 9406 3077
frank(at)fmp-berlin.de

Chemical Systems Biology (Ronald Frank)

Calcium-loaded Calmodulin in the ligand free (above) state and in complex with a target peptide, showing the large conformational changes of CaM during target binding (taken from Hultschig et al., 2004, J. Mol. Biol. 343, 559-568).

Biology focus

The general aim of the group is the development of approaches for the directed modulation of complex regulatory networks as a basis for the discovery of novel principles for pharmacological interventions. Stimulated by earlier work of the principle investigator, we have chosen the large group of proteins regulated by calcium/calmodulin (CaM). CaM is a small acidic ubiquitous protein (∼148 amino acids) and conserved extraordinarily throughout the evolution of eukaryotes. Essentially all vertebrates contain identical CaMs, even though encoded by different genes. CaM is an integral modulator of many calcium-dependent processes in virtually every eukaryotic cell type. It functions as a cytosolic calcium receptor and binds up to four calcium ions with very high affinity. In this way it responds to a variety of different extra cellular signals which increase the cytosolic calcium level. Thereby CaM decodes the Ca2+ signal that is brought about by the influx of Ca2+ through respective Ca-channels in the plasma membrane and activates or deactivates both kinases and phosphatases. Furthermore, calmodulin regulates enzymes involved in the signal transduction such as cyclic nucleotide phospodiesterases, adenylate cyclases, nitric oxide synthetase and plasma membrane calcium ATPases. There are, however, protein families including neuromodulin, myosin, ionchannels that bind CaM at remarkably low concentrations or even in the absence of calcium.

Upon loading the four calcium-binding sites with calcium ions, a conformational change in CaM is rapidly induced resulting in the exposition of hydrophobic patches on its surface, which enable CaM to bind to a variety of different target proteins with extraordinary high affinity (see Figure, right). A number of organic hydrophobic molecules such as derivatives of naphthalene and natural peptides such as peptide hormones, neurotransmitters and venoms also bind CaM with high affinity.

For the first part of our calmodulin project we have scanned all protein components of the human ribosome in the format of 21mer overlapping peptide fragments (5000 peptides altogether, including reference peptides and proteins) for CaM binding. We have confirmed many sites reported in the literature and have identified several new ones. Functional analysis of these regulatory sites is underway.

 

Technical steps in the preparation of chemical microarrays following the SC2 process: (a) parallel SPOT synthesis of a combinatorial library of compounds; (b) punching out spots and dissolve the cellulose matrix in Acid, followed by precipitation, washing and dissolution in DMSO; (c) printing the microarrays by transfer of nL aliquots onto a prepared glass slide; (d) probing with fluorescently labelled target.

Chemistry focus

With the aim of exploring molecular scaffolds for their utility as probes for the modulation of our target proteins, our research program involves the chemical synthesis of such interesting scaffolds (mainly based on peptides and low molecular weight small molecules), the adaptation of their synthetic routes to our special chemical microarray technology, the preparation of combinatorial libraries around such scaffolds and their biological testing (see Figure, right).

We have implemented all technical steps for the production of chemical libraries in the format spatially immobilized microarrays. A new library of almost 2.500 diketopiperazines was successfully obtained and will be tested for specific CaM binders. This generic binder library is now available to probe many other target proteins.

Libraries of soluble compounds are prepared to complement the chemical collection of the FMP in regions of low populated chemical space. A synthetic route to tetra- and penta-substituted dihydropyrroles (Zhu et al., J. Comb. Chem. 2009) has been established and analogues are being produced.

Part of the work of our group is to support the drug research efforts of the institute with new technologies and resources.

Leibniz-Institut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de