OBSERVATION VIA ONE-DIMENSIONAL C-13(ALPHA) NMR OF LOCAL CONFORMATIONAL SUBSTATES IN THERMAL UNFOLDING EQUILIBRIA OF A SYNTHETIC ANALOG OF THE GCN4 LEUCINE-ZIPPER

Synthesis of a 33-residue, capped leucine zipper analogous to that in GCN4 is reported. Histidine and arginine residues are mutated to lysin e to reduce the unfolding temperature. CD and ultracentrifugation stud ies indicate that the molecule is a two-stranded coiled coil under ben ign conditions. Versions of the same peptide are made with 99% C-13(al pha) at selected sites. One-dimensional C-13 NMR spectra are assigned by inspection and used to study thermal unfolding equilibria over the entire transition from 8 to 73 degrees C. Spectra at the temperature e xtremes establish the approximate chemical shifts for folded and unfol ded forms at each labeled site. Resonances for each amino acid appear at both locations at intermediate T, indicating that folded and unfold ed forms interconvert slowly (much greater than 2 ms) on the NMR time scale. Moreover, near room temperature, the structured form's resonanc e is double at several, but not all, sites, indicating at least two sl owly interconverting, structured, local conformational substates. Anal ysis of the dynamics is possible. For example, near room temperature a t the Val-9, Ala-24, and Gly-31 positions, the equilibrium constant fo r interconversion of the two structured forms is near unity and the ti me scale is greater than or equal to 10-20 ms.