Myoglobin and Apomyoglobin in their Native, Molten Globule and Acid-Denaturated States. A Dielectric Relaxation Study

Abstract

Abstract The acid-induced unfolding of myoglobin and its haem-free derivative apo-myoglobin is studied by broadband dielectric relaxation spectroscopy at pH values near 5, 4 and 2, which reflect the transition N → MG → UA from the native state (N) via a molten globule state (MG) to an acid-unfolded state (UA). Spectral changes are singled out by considering difference spectra for the transitions N → MG and N → UA. A pronounced increase of the amplitude of the tumbling motion of holo-myoglobin and apo-myoglobin in the sequence N → MG→ UA reflects the increase of their electrical dipole moments by progressive unfolding. A distinct high-frequency shoulder of the tumbling mode indicates an enhanced internal protein dynamics in the unfolded states. The calculated Stokes radii confirm a compact nature of the MG state and a large increase in size of the UA state relative to the native state. In the native state the Stokes radii deduced from the dielectric spectra agree fairly well with those deduced from dynamic light scattering. In the MG state, and particularly in the UA state, the Stokes radii determined by light scattering are markedly larger than those determined by dielectric relaxation. The difference is attributed to aggregation of the unfolded species, which affects dynamic light scattering more than dielectric relaxation. In the high-frequency regime, the dielectric spectra indicate that unfolding enhances the number of water molecules showing bulk-like dynamics, but the effect is rather weak.