A fluorescent hinge peptide for the dimerization of proteins through disulfide formation

• Applicant: Professor Dr. Armin Geyer
• Subject Area: Biological and Biomimetic Chemistry ; Organic Molecular Chemistry - Synthesis and Characterisation
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 427497450

We recently described a disulfide-bridged dimeric peptide which forms through oxidative folding of the 12 amino acids motif CX3CX2CX3C (C = cysteine, X = other amino acids). The high cysteine content – every third amino acid is cysteine – exceeds that of natural cysteine motifs and results in a unique homodimerization selectivity upon air oxidation. Not only the isolated peptide undergoes covalent dimerization but also proteins up to a size of 150 kDa that are fused to the peptide by recombinant DNA technology. One of the homodimeric peptide shows blue fluorescence that is probably caused by the excimer formation between its two tryptophans. We will investigate the structural requirements of this effect that increases the application potential of this hinge dimers as fluorescence tags. The molecular requirements of the dimerization process and the oxidative folding will be studied by systematic structural modifications. We will try to identify intermediates of the balance act of oxidative folding of eight cysteines by complementary synthetic and analytic strategies. The structural requirements of the dimerization process will be tested by systematic amino acid variations at the cysteines, the turn amino acids, and the side chain charges with a special focus on the heterodimeric peptides. Spectroscopic methods will be used to characterize the conformational mobility of the two halves of the hinge from its response to systematic variations of the structural environment i.e. the “loading” of the hinge with polypeptides of different size. The central aim of this application is to identify the structural prerequisites of the tetradisulfide dimeric peptide for its high tolerance to different protein loads and its unique regioselectivity of oxidative folding. The investigation of physicochemical properties, conformation, and cooperative mobility of this novel protein dimerization motif will be of interest for peptide/protein folding and stability in general.

• DFG Programme: Research Grants