Temperature dependence of intramolecular dynamics of the basic leucine zipper of GCN4: implications for the entropy of association with DNA.

The basic leucine zipper domain of the yeast transcription factor GCN4 consists of a C-terminal leucine zipper and an N-terminal basic DNA-binding region that achieves a stable structure only after association with DNA. Backbone dynamics of a peptide encompassing the basic and leucine zipper bZip domain (residues 226-281) are described using NMR spectroscopy. The 15N longitudinal relaxation rates, 15N transverse relaxation rates, and {1H}-15N nuclear Overhauser effects were measured for the backbone amide nitrogen atoms at 290 K, 300 K, and 310 K. The relaxation data were interpreted using reduced spectral density mapping to determine values of the spectral density function, J(omega), at the frequencies 0, omegaN, and 0.87omegaH to characterize overall and intramolecular motions on picosecond-nanosecond timescales. To account for the temperature dependence of overall rotational diffusion, the J(0) values were normalized using Stoke's Law. At 310 K, the 13Calpha and 13CO chemical shifts in conjunction with the spectral density values indicate that the leucine zipper sequence forms a highly ordered alpha-helix, while the basic region populates an ensemble of highly dynamic transient structures with substantial helical character. The normalized values of J(0) and the values of J(0.87omegaH) for residues in the leucine zipper dimerization domain are independent of temperature. In contrast, residues in the basic region exhibit pronounced increases in the normalized J(0) and decreases in J(0.87omegaH) as temperature is decreased. A strong correlation exists between the temperature dependence of 13CO chemical shifts and of J(0.87omegaH). These results suggest that, for the basic region, lowering the temperature increases the population of transient helical conformations, and concomitantly reduces the amplitude or timescale of conformational fluctuations on picosecond-nanosecond timescales. Changes in the conformational dynamics of the peptide backbone of the basic region that accompany DNA binding contribute to the overall thermodynamics of complex formation. The change in backbone conformational entropy derived from NMR spin-relaxation data agrees well with the result calculated from calorimetric measurements. Restriction of the conformational space accessible to the basic region may significantly reduce the entropic cost associated with formation of the basic region helices consequent to DNA binding. Copyright 1999 Academic Press Limited.