%0 Generic
%A Pham, Truc Lam
%C Heidelberg
%D 2024
%F heidok:35552
%R 10.11588/heidok.00035552
%T Design of metal-binding globular β-sheet miniproteins as biosensors and artificial enzymes
%U https://archiv.ub.uni-heidelberg.de/volltextserver/35552/
%X In recent years, numerous proteins with novel structures and functions have been created through design. However, these are mostly α-helical structures whose sequence-structure relationship are largely understood. This is not the case for globular β-sheet proteins.  In the present work, the well-studied WW domain of hPin1 and the β-hairpin Trpzip2 were selected as scaffolds to design mini-metalloproteins for catalytic and biosensory applications. For this purpose, a binding site of three histidines was introduced on the surface of the β-sheets, as well as additional mutations to influence the stability of the peptides and avoid additional binding sites.  The designed WW domains selectively bound Ni(II), Cu(II) and Zn(II) with characteristic reversible conformational changes. Metal binding led to an increase in thermostability, with the metal complexes displaying melting temperatures of up to 70 °C. Furthermore, a positive correlation between the thermostability of the apo-peptide and the binding affinities to the metal ions was observed. The designed β-hairpin behaved similarly to the WW domains in its binding properties but displayed even higher thermostabilities of up to 80 °C, as well as stability towards chaotrophic salts and organic solvents.  The mini-metalloproteins were only slightly to moderately catalytically active (hydrolysis, phenol oxidation and CuAAC), but their selective and reversible conformational change makes them very suitable for biosensing. By introducing an artificial fluorescent amino acid with a 7-mercapto-4-methylcoumarin side chain via late-stage functionalization on the solid phase, an iFRET-based metal ion sensor was developed that also detected bioactive molecules such as glyphosate or pyrophosphate through the competition of metal binding or direct interaction via the change in FRET intensity. An expansion of the analyte scope was achieved by the design of a receptor array of mini-metalloproteins, which was able to distinguish glyphosate from other bioactive molecules.  Solid phase peptide synthesis was used to synthesize the miniproteins. However, the amino acid aspartate, which may be important for coordination sites and stability, can lead to undesired aspartimide formation during synthesis. To suppress this side reaction, the use of the cyanosulfurylide protecting group (CSY) was optimized for microwave-assisted synthesis. First, the CSY-protected aspartate building block was synthesized on a multi-gram scale. The coupling of the building block was carried out under heating, but the deprotection of the temporary protecting group by piperidine must be carried out at room temperature. The CSY-protected peptides were obtained in excellent quality were finally deprotected under oxidative conditions, which required the addition of hexafluoroisopropanol.