TY - JOUR
T1 - Site-specific experiments on folding/unfolding of Jun coiled coils
T2 - Thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine-18
AU - D'Avignon, D. André
AU - Bretthorst, G. Larry
AU - Holtzer, Marilyn Emerson
AU - Schwarz, Kathleen A.
AU - Angeletti, Ruth Hogue
AU - Mints, Lisa
AU - Holtzer, Alfred
PY - 2006/10/15
Y1 - 2006/10/15
N2 - The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 α-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil aimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate constants for GCN4 as determined by chevron analysis and NMR in differing temperature regimes.
AB - The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 α-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil aimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate constants for GCN4 as determined by chevron analysis and NMR in differing temperature regimes.
KW - Coiled coils
KW - Leucine zipper
KW - Protein denaturation
KW - Protein folding
KW - Spin inversion transfer
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U2 - 10.1002/bip.20555
DO - 10.1002/bip.20555
M3 - Article
C2 - 16767740
AN - SCOPUS:33749326385
SN - 0006-3525
VL - 83
SP - 255
EP - 267
JO - Biopolymers
JF - Biopolymers
IS - 3
ER -