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Designed to be stable: crystal structure of a consensus ankyrin repeat protein
Andreas Kohl1, H Kaspar Binz, Patrik Forrer
1Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Summary
Scientists engineered a stable protein scaffold based on ankyrin repeats (AR) for creating new binding molecules. This versatile AR protein fold can be customized for diverse molecular interactions.
Area of Science:
- Protein Engineering
- Structural Biology
- Biochemistry
Background:
- Ankyrin repeat (AR) proteins are crucial for diverse protein-protein interactions across all life forms.
- Their inherent ability to mediate interactions suggests potential for designing novel binding molecules.
Purpose of the Study:
- To design and characterize a consensus ankyrin repeat (AR) protein scaffold.
- To evaluate its stability, expression, and suitability for creating customized binding molecules.
Main Methods:
- Sequence and structural analysis to design a consensus AR repeat with fixed framework and randomized interacting residues.
- Generation of combinatorial AR protein libraries with varying numbers of consensus repeats.
- Expression in E. coli, purification, and determination of crystal structure to 2.0-A resolution.
Main Results:
- Designed AR proteins expressed highly (up to 30% of total protein) and stably in E. coli.
- Crystal structure revealed a well-defined fold with complementary hydrophobic interfaces and regular hydrogen bonding networks.
- Glycine residues were consistently found in turn regions, contributing to structural regularity.
Conclusions:
- The consensus AR domain fold is an intrinsically stable and well-expressed scaffold.
- It effectively displays randomized interacting residues, making it an excellent basis for designing novel binding molecules.