eprintid: 35549
rev_number: 12
eprint_status: archive
userid: 8514
dir: disk0/00/03/55/49
datestamp: 2024-10-30 13:31:07
lastmod: 2024-11-04 12:40:05
status_changed: 2024-10-30 13:31:07
type: doctoralThesis
metadata_visibility: show
creators_name: Häge, Florian Raphael
title: Design of Structured Adhesion Miniproteins for Tissue Engineering
subjects: ddc-540
divisions: i-120100
adv_faculty: af-12
abstract: of 55 ± 0.5 °C and bound Ca(II) with a Kd = 7.9 ± 3.4 μM. The grafting approach was used to incorporate Ca(II)-binding EF-hand domains into the loops and N-termini of miniprotein scaffolds. [6, 9] EF-hand 2 of Calmodulin was grafted onto the N-terminus of the Trp-cage designed by Neidigh et al. in the Ca(II)-binding model Calmcage. [10] The fusion miniprotein folded into a similar structure to its parent peptide with an increased thermostability Tm = 50 ± 3 °C. Calmcage bound Tb(III) with a Kd = 200 ± 40 μM and Ca(II) with a Kd = 64 ± 26 mM. Another successfully grafted design was created from the EF-hand 3 of Calmodulin and the β-hairpin system C4 by Anderson et al. in the model Calmzip3. [11] It folded into a similar-to-native structure and changed conformation upon Ca(II)-binding with a thermostability of Tm = 47.5 ± 1.0 °C for the holo- and Tm = 47 ± 3 °C for the apo-protein. Calmzip3 revealed strong Tb(III) binding with a Kd = 0.96 ± 0.15 μM and Ca(II) binding in the same order of magnitude as the weakest value for Calmodulin and the strongest value for Laminin with Kd = 3.9 ± 1.4 μM.
The computational design of a Superoxide Dismutase (SOD) site on the scaffold of the SH3 domain resulted in the model SO1. It folded into a β-sheet structure with high thermostability that increased in the presence of bivalent transition metals from Tm = 47 ± 0.5 °C for the holo- to Tm = 74 ± 1 °C for the Ni(II)-bound state. It showed SOD activity in the presence of Cu(II) in the same order of magnitude as small organic Cu(II)-complex mimics, outcompeting all miniprotein SOD enzymes to date with an apparent rate constant kSOD = 6.14 ± 0.58 × 107 M-1 s-1. [12-16] It therefore presented itself not only as a proof of concept for computational design but also as a potential model for studying SOD enzymes.
In conclusion, all three approaches delivered functional miniproteins with Ca(II)-binding miniproteins that range from millimolar to low micromolar Kd-values. With binding affinities in the same range, they serve as ideal candidates for Ca(II)-dependent carbohydrate recognition after the example of D-type lectins.
date: 2024
id_scheme: DOI
id_number: 10.11588/heidok.00035549
ppn_swb: 1907470913
own_urn: urn:nbn:de:bsz:16-heidok-355495
date_accepted: 2024-08-23
advisor: HASH(0x55e83b1fb3b0)
language: eng
bibsort: HAGEFLORIADESIGNOFST20241025
full_text_status: public
place_of_pub: Heidelberg
citation:   Häge, Florian Raphael  (2024) Design of Structured Adhesion Miniproteins for Tissue Engineering.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/35549/1/dissertation_full.pdf