Native and non-native secondary structure and dynamics in the pH 4 intermediate of apomyoglobin

The partly folded state of apomyoglobin at pH 4 represents an excellent mod el fur an obligatory kinetic folding intermediate. The structure and dynami cs of this intermediate state have been extensively examined using NMR spec troscopy. Secondary chemical shifts, H-1-H-1 NOEs, and amide proton tempera ture coefficients have been used to probe residual structure in the interme diate state, and NMR relaxation parameters T-1 and T-2 and (H-1)-N-15 NOE h ave been analyzed using spectral densities to correlate motion of the polyp eptide chain with these structural observations. A significant amount of he lical structure remains in the pH 4 state, indicated by the secondary chemi cal shifts of the C-13(alpha), (CO)-C-13, H-1(alpha), and C-13(beta) nuclei , and the boundaries of this helical structure are confirmed by the locatio ns of H-1-H-1 NOEs. Hydrogen bonding in the structured regions is predomina ntly nativelike according to the amide proton chemical shifts and their tem perature dependence. The locations of the A, G, and H helix segments and th e C-terminal part of the B helix are similar to those in native apomyoglobi n, consistent with the early, complete protection of the amides of residues in these helices in quench-flow experiments. These results confirm the sim ilarity of the equilibrium form of apoMb at pH 4 and the kinetic intermedia te observed at short times in the quench-flow experiment. Flexibility in th is structured core is severely curtailed compared with the remainder of the protein, as indicated by the analysis of the NMR relaxation parameters. Re gions with relatively high values of J(0) and low values of J(750) correspo nd well with the A, B, G, and H helices, an indication that nanosecond time scale backbone fluctuations in these regions of the sequence are restricte d. Other parts of the protein show much greater flexibility and much reduce d secondary chemical shifts. Nevertheless, several regions show evidence of the beginnings of helical structure, including stretches encompassing the C helix-CD loop, the boundary of the D and E helices, and the C-terminal ha lf of the E helix. These regions are clearly not well-structured in the pH 4 state, unlike the A, B, G, and H helices, which form a nativelike structu red core. However, the proximity of this structured core most likely influe nces the region between the B and F helices, inducing at least transient he lical structure.