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Improved Stability of the Jun-Fos Activator Protein-1 Coiled A STOPPED-FLOW CIRCULAR DICHROISM KINETIC ANALYSIS*
 

Summary: Improved Stability of the Jun-Fos Activator Protein-1 Coiled
Coil Motif
A STOPPED-FLOW CIRCULAR DICHROISM KINETIC ANALYSIS*
Received for publication,March 2, 2007, and in revised form, May 2, 2007 Published, JBC Papers in Press,May 4, 2007, DOI 10.1074/jbc.M701828200
Jody M. Mason1
, Urs B. Hagemann, and Katja M. Arndt2
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 (JunWPh1). Enriched winners differ
from each other in only two of ten semi-randomized positions,
with Tm values of 28 °C and 37 °C over wild-type. cFos-JunW,
cFos-JunWPh1, and two intermediate mutants (cFos-JunWQ21R
and cFos-JunWE23K) 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 expo-
nential. In contrast, in the unfolding direction, all molecules

  

Source: Arndt, Katja - Institut für Biologie III, Albert-Ludwigs-Universität Freiburg

 

Collections: Biotechnology; Biology and Medicine