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J. Biol. Chem., Vol. 282, Issue 32, 23015-23024, August 10, 2007

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Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif

A STOPPED-FLOW CIRCULAR DICHROISM KINETIC ANALYSIS^*

From the Institute of Biology III, Albert-Ludwigs University of Freiburg, Schaenzlestrasse 1, D-79104 Freiburg, Germany

Two c-Jun leucine zipper variants that bind with high affinity to c-Fos have been selected using semirational design combined with protein-fragment complementation assays (JunW) or phage display selection (JunW_Ph1). Enriched winners differ from each other in only two of ten semi-randomized positions, with T_m values of 28 °C and 37 °C over wild-type. cFos-JunW, cFos-JunW_Ph1, and two intermediate mutants (cFos-JunW_Q21R and cFos-JunW_E23K) display biphasic kinetics in the folding direction, indicating the existence of a folding intermediate. The first reaction phase is fast and concentration-dependent, showing that the intermediate is readily populated and dimeric. The second phase is independent of concentration and is exponential. In contrast, in the unfolding direction, all molecules display two-state kinetics. Collectively this implies a transition state between unfolded helices and dimeric intermediate that is readily traversed in both directions. We demonstrate that the added stability of cFos-JunW_Ph1 relative to cFos-JunW is achieved via a combination of kinetic rate changes; cFos-JunW_E23K has an increased initial dimerization rate, prior to the major transition state barrier while cFos-JunW_Q21R displays a decreased unfolding rate. The former implies that improved hydrophobic burial and helix-stabilizing mutations exert their effect on the initial, rapid, monomer-collision event. In contrast, electrostatic interactions exert their effect late in the folding pathway. Although our focus is the leucine zipper region of the oncogenic transcription factor Activator Protein-1, coiled coils are ubiquitous and highly specific in their recognition of partners. Consequently, generating kinetic-based rules to predict and engineer their stability is of major significance in peptide-based drug design and nano-biotechnology.

Received for publication, March 2, 2007 , and in revised form, May 2, 2007.

^* This work was supported by Grant Ar 373/1-1, 1-2 from the Emmy Noether Program of the Deutsche Forschungs Gemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Inst. of Biology III, Albert-Ludwigs University of Freiburg, Schaenzlestr 1, D-79104 Freiburg, Germany. Tel.: 49-761-203-2748-2763; Fax: 49-761-203-2745; E-mail: jody{at}biologie.uni-freiburg.de. ² To whom correspondence may be addressed: Inst. of Biology III, Albert-Ludwigs University of Freiburg, Schaenzlestr 1, D-79104 Freiburg, Germany. Tel.: 49-761-203-2748/2763; Fax: 49-761-203-2745; E-mail: katja{at}biologie.uni-freiburg.de.

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