A folding pathway for βpep-4 peptide 33mer: From unfolded monomers and β-sheet sandwich dimers to well-structured tetramers

It was recently reported that a de novo designed peptide 33mer, βpep- 4, can form well-structured β-sheet sandwich tetramers (Ilyina E, Roongta V, Mayo KH, 1997b, Biochemistry 36:5245-5250). For insight into the pathway of βpep-4 folding, the present study investigates the concentration dependence of βpep-4 self-association by using ¹H-NMR pulsedfield gradient (PFG)-NMR diffusion measurements, and circular dichroism. Downfield chemically shifted αH resonances, found to arise only from the well-structured βpep-4 tetramer state, yield the fraction of tetramer within the oligomer equilibrium distribution. PFG-NMR-derived diffusion coefficients, D, provide a means for deriving the contribution of monomer and other oligomer states to this distribution. These data indicate that tetramer is the highest oligomer state formed, and that inclusion of monomer and dimer states in the oligomer distribution is sufficient to explain the concentration dependence of D values for βpep-4. Equilibrium constants calculated from these distributions [2.5 x 10⁵ M^-1 for M-D and 1.2 x 10⁴ M^-1 for D-T at 313 K] decrease only slightly, if at all, with decreasing temperature indicating a hydrophobically mediated, entropy-driven association/folding process. Conformational analyses using NMR and CD provide a picture where 'random coil' monomers associate to form molten globule-like β-sheet sandwich dimers that further associate and fold as well-structured tetramers. βpep-4 folding is thermodynamically linked to self-association. As with folding of single- chain polypeptides, the final folding step to well-structured tetramer βpep- 4 is rate limiting.