NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. T he conformational propensities and polypeptide backbone dynamics of this st ate have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance as signment strategy tailored to low protein concentration (0.2 mM) and poor c hemical shift dispersion. An estimate of the population and location of res idual secondary structure has been made by examining deviations of C-13(alp ha), (CO)-C-13, and H-1(alpha) chemical shifts from random coil values, sca lar (3)J(HN),(H alpha) coupling constants and H-1-H-1 NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences , provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN),(H alpha) coupling constants are poor diagnostics of secondary structure formation. Substantia l populations of helical structure, in dynamic equilibrium with unfolded st ates, are formed in regions corresponding to the A and H helices of the fol ded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide bac kbone dynamics of acid-unfolded apomyoglobin have been investigated using r educed spectral density function analysis of N-15 relaxation data. The spec tral density J(omega (N)) is particularly sensitive to variations in backbo ne fluctuations on the picosecond to nanosecond time scale. The central reg ion of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega (N)) Of restricted motions on the picosecond to n anosecond time scale in the A and H helix regions where there is a propensi ty to populate helical secondary structure in the acid-unfolded state. Back bone fluctuations are also restricted in parts of the B and G helices due t o formation of local hydrophobic clusters. Regions of restricted backbone f lexibility are generally associated with large buried surface area. A signi ficant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably aris e from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyogl obin formed at pH 2.3 is an excellent model for the events that are expecte d to occur in the earliest stages of protein folding, providing insights in to the regions of the polypeptide that spontaneously undergo local hydropho bic collapse and sample nativelike secondary structure.