Conformational and dynamic characterization of the molten globule state ofan apomyoglobin mutant with an altered folding pathway

Kinetic and equilibrium studies of apomyoglobin folding pathways and interm ediates have provided important insights into the mechanism of protein fold ing. To investigate the role of intrinsic helical propensities in the apomy oglobin folding process, a mutant has been prepared in which Asn132 and Glu 136 have been substituted with glycine to destabilize the H helix. The stru cture and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone C-13(alp ha) and (CO)-C-13 chemical shifts from random coil values reveal high popul ations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to th e wild-type molten globule. Heteronuclear {H-1}-N-15 NOES show that, althou gh the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange me asurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helic es and the CD region. The kinetic and equilibrium molten globule intermedia tes formed by N132G/EI36G are similar in structure. Although both the wild- type apomyoglobin and the mutant fold via compact helical intermediates, th e structures of the intermediates and consequently the detailed folding pat hways differ. Apomyoglobin is therefore capable of compensating for mutatio ns by using alternative folding pathways within a common basic framework. T ertiary hydrophobic interactions appear to play an important role in the fo rmation and stabilization of secondary structure in the H helix of the N132 G/ E136G mutant. These studies provide important insights into the interpla y between secondary and tertiary structure formation in protein folding.