Improved Repeat Protein Stability by Combined Consensus and Computational Protein Design

Abstract

High protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N-and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap.

Unexpected dynamics in femtomolar complexes of binding proteins with peptides

“…4 ), and affinity determination by FP resulted in a highly similar K d to the peptide (KR) 5 : 36.1 ± 2.9 nM for N Y M 4 C versus 30.5 ± 2.3 nM for N A M 4 C (mean ± S.D.) 14 . This combined evidence clearly demonstrates that the two protein variants respond almost identically to peptide binding, and data below therefore mostly describe structures employing the new N-cap.…”

“…Biochemical data indicated that the original y east-derived N Y -terminal cap possesses an intrinsic instability against E. coli proteases, likely due to insufficient packing of the cap against the M1 module. The redesigned N-cap, described in Michel et al 14 and referred to as N A ( a rtificial), displayed improved stability and yielded very similar CSPs. A comparison of CSPs between N Y M 4 C and N A M 4 C upon binding (KR) 4 revealed similar binding for both constructs (Supplementary Fig.…”

“…Finally, we describe binding of (KR) n to NM x C with n = 4, 5, 6 and 7 and x = 4 and 7. We looked at two versions of the proteins with different N-caps, the so-called yeast-derived N Y cap 6 , 13 and the recently developed more stable N A cap 14 . To accomplish the challenging task of chemical shift assignments in proteins composed of a repetitive amino acid sequence we applied segmental isotope labeling using expressed protein ligation.…”

“…Coordinates of unliganded (generated in a previous study 14 ) and (KR) 4 -bound N A M 4 C (generated in this study) were deposited in the PDB database under accession codes 7R0R [10.2210/pdb7R0R/pdb] and 8OH7 [10.2210/pdb8OH7/pdb], respectively. Chemical shifts for the diamagnetic references were deposited for the complex with (KR) 4 on the BMRB database under codes 51290 , 51291 , and 51292 for the three different attachment sites of Tm-tagged proteins.…”

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst

“…Conserved amino acids may replace non-conserved amino acids to obtain more stable variants ( Steipe, 1999 ; Porebski and Buckle, 2016 ). This method has been proven relatively reliable for improving protein thermostability ( Wojcik et al, 2019 ; Hayashi et al, 2022 ; Michel et al, 2023 ). Sequences whose query identity was above 30% and below 90% are screened for the alignment of the T7RNAP homologs ( Bendl et al, 2016 ).…”