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Folding of a designed simple ankyrin repeat protein

Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland

(RECEIVED June 17, 2004; FINAL REVISION August 2, 2004; ACCEPTED August 5, 2004)

Ankyrin repeats (AR) are 33-residue motifs containing a -turn, followed by two -helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5°C both follow monoexponential kinetics with k_folding = 28 sec^–1 and k_unfolding = 0.9 sec^–1. Kinetic and equilibrium unfolding parameters at 5°C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.

Keywords: ankyrin repeat; calorimetry; protein design; protein folding; protein stability

Reprint requests to: Ilian Jelesarov, Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; e-mail: iljel{at}bioc.unizh.ch; fax: ++41-1-635-6805.

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