In vivo photo-regulation of protein structure and function by genetically encoded photoswitchable-click amino acids

• Applicant: Dr. Christian Hoppmann
• Subject Area: Biochemistry
• Term: from 2012 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 211375240

Aim of the proposed project is the in vivo modification of proteins to control their biological activity by light. Therefore newly designed photo-switchable-click amino acids (PSCaa) will be introduced site-specifically into the protein backbone by evolved orthogonal tRNA/synthetase pairs. PSCaa contain an azobenzene switch element equipped with a light-activatable vinyl group that will enable selective thiol-ene click reaction with opportunely distanced cysteine residues, thus building photo-switchable bridges that will stabilize or destabilize protein folding - and thereby alter protein function - depending on their cis/trans status. The higher selectivity of this method comparing to the current methodology, the post-translational modification of proteins containing two cysteines by using thiol-reactive azobenzene switches is of advantage because the application of the current method in living systems is limited by the variety of built products. For biologically active polypeptides we could already show that after chemical incorporation of a PSCaa an intramolecular bridge to a neighboring cysteine was built specifically and that this photo-controlled bridge allowed conformation and biopotency of the polypeptide to be controlled by light. We will proof the feasibility of this approach on calmodulin (CaM), a eukaryotic regulatory protein showing dramatic conformational changes between inactive and active state. At first, for the novel PSCaa new specific orthogonal sets of tRNA/tRNA-synthetase pairs will be evolved and PSCaa-Cys bridges will be formed in vitro on CaM mutants isolated from E. coli. For a set of differently bridged mutants we will estimate binding affinities to epitopes of known calmodulin-binding proteins. The mutant showing the highest affinity change upon photo-switching will be imported into mammalian cells to study specific CaM functions in vivo through light-control.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Lei Wang, Ph.D.