7R0R

Solution structure of the designed Armadillo repeat protein N(A4)M4C(AII) refined by pseudocontact shifts

Summary for 7R0R

• Entry DOI: 10.2210/pdb7r0r/pdb
• Related: 7QNP
• NMR Information: BMRB: 34707,51290,51291,51292
• Descriptor: Designed Armadillo Repeat Protein N(A4)M4C(AII) (1 entity in total)
• Functional Keywords: repeat protein, designed armadillo repeat protein, peptide binding protein
• Biological source: synthetic construct
• Total number of polymer chains: 1
• Total formula weight: 61907.22

Authors

Cucuzza, S.,Zerbe, O. (deposition date: 2022-02-02, release date: 2022-06-22, Last modification date: 2024-06-19)

Primary citation

Michel, E.,Cucuzza, S.,Mittl, P.R.E.,Zerbe, O.,Pluckthun, A.
Improved Repeat Protein Stability by Combined Consensus and Computational Protein Design.
Biochemistry, 62:318-329, 2023

PubMed 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.

PubMed: 35657362
DOI: 10.1021/acs.biochem.2c00083

Experimental method

SOLUTION NMR