A fluorescent hinge peptide for the dimerization of proteins through disulfide formation
Organic Molecular Chemistry - Synthesis and Characterisation
Final Report Abstract
A cysteine-rich peptide, which dimerises regioselectively in the presence of atmospheric oxygen, formed the preparatory work for this project. When linked to other peptides or even proteins, it can be used as a domain for the covalent linkage of different loadings. In contrast to other molecular dimerisation techniques, this 12mer peptide forms two covalent intermolecular bonds, which makes it comparable to the hinge region of an Ig antibody and can even replace the antibody hinge (also preliminary work). In the initial work on this project, computer modelling - metadynamics - was used to identify the dominant degrees of freedom of movement of this peptide hinge. The focus of the preparative part of this project was the characterisation of the molecular requirements for the self-recognition of the 12mer peptide as a prerequisite for air oxidation to the 24mer hinge. Tryptophan (Trp) plays a crucial role in this process, which was underlined by the unusual quenching of Trp fluorescence upon dimerisation. The hinge peptide was assembled with differently substituted Trp analogues and the fluorescence quenching was quantified. Although some of the amino acids were chosen because they show stronger fluorescence than Trp, none showed stronger relative fluorescence quenching between 12mer tetrathiol and 24mer hinge than the natural amino acid Trp. No amino acid has yet been identified that shows a more pronounced second emission maximum in visible light than that observed for Trp in the 24mer hinge. The conformational equilibrium of the internal motion of the 24mer hinge is accelerated in some analogues to such an extent that new data for further modelling could be obtained from their high-resolution NMR spectra. A relevant component of the project was the establishment of collaborations with other working groups in order to develop new applications for the hinge peptide. To this end, a collaboration was initiated in which receptor dimerisation is to be triggered by a dimeric ligand. A cooperation on structure elucidation in the solid state was also initiated. The basis for this was the idea of linking with a further codable dimerisation domain, whose crystal structure has already been well investigated, in order to obtain a co-crystal with the 24mer hinge. This is also an important prospect from this project. Unexpectedly, the natural amino acid Trp showed the strongest fluorescence quenching of all the examples investigated and the bathochromic shift of the maximum and the increase in fluorescence intensity by suitable substituents led to reduced relative fluorescence quenching. These are important data for the investigation of further structural variations synthesised using the technique developed in this project.
Publications
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Comparing the Hinge-Type Mobility of Natural and Designed Intermolecular Bi-disulfide Domains. Frontiers in Chemistry, 8.
Horx, Philip & Geyer, Armin
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Defining the mobility range of a hinge-type connection using molecular dynamics and metadynamics. PLOS ONE, 15(4), e0230962.
Horx, Philip & Geyer, Armin
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ORCHEM 2022, 05.09.2022 - 07.09.2022, Münster, Cross Coupling of Indoles for the Systematic Variation of the Electron Density in Tryptophan
André Schmiegel
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16th German Peptide Symposium, 28.08.2023 - 30.08.2023, Jena, Synthesis of Fluorescent - Amino Acids and Peptides by Palladium-Catalyzed Coupling Methods
Lorenz Rau
