Improved stability of the Jun-Fos Activator Protein-1 coiled coil motif: A stopped-flow circular dichroism kinetic analysis.
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 DeltaT(m) values of 28 degrees C and 37 degrees 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.
Mason
Jody M
JM
Institute of Biology III, Albert-Ludwigs University of Freiburg, Schaenzlestrasse 1, D-79104 Freiburg, Germany. jody@biologie.uni-freiburg.de
Hagemann
Urs B
UB
Arndt
Katja M
KM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry*; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry*; D013816:Thermodynamics; D018808:Transcription Factor AP-1 / Q000737:chemistry*; D014157:Transcription Factors / Q000378:metabolism
D010455:Peptides; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014157:Transcription Factors