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

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.

Molecular Insights into Sequence-Specific Protein Hydrolysis by a Soluble Zirconium-Oxo Cluster Catalyst

“…At pH 4.5 (1 mM acetic acid) all of the spectra remained unchanged, indicating that the structure of Mb is largely preserved. Because partial unfolding of Mb into a molten globule state at pH <5 has been reported before, 60 the concentration of acetic acid was further increased. However, even at pH 3.4 (5 mM acetic acid), the changes in the spectra were insignificant compared to the changes induced by 10 equiv of Zr 6 (100 μM), corroborating the need for Zr 6 to reach a similar state of protein unfolding.…”

Unveiling GruPol: Predicting Electric and Electrostatic Properties of Macromolecules via the Building Block Approach

“…38,43−46 Consequently, these variations impact folding, solubility, or reactivity. 46 The influence on protein folding is directly observable in Figure 5, where titratable residues with opposite charges tend to approach each other, thus directly affecting the dipole moment. Naturally, the presence of local charges depends on the pH of the solution and the pK a of each residue.…”

Nonlinear dielectric response of dilute protein solutions

“…11 The gap in eld magnitudes between bulk liquids and protein solutions suggests that NDE can be used to probe the protein component separately from a much weaker nonlinear response of the surrounding solvent. Given that the dipole moment is sensitive to protein's conformations and physiological activity, [12][13][14][15][16] NDE can potentially monitor alterations in the protein structure, phosphorylation, and redox reactions. Despite some preliminary reports on cells 17,18 and membrane-bound proteins, 19 NDE of proteins in solution has not been measured and the formalism proposed here remains a theoretical prediction at this moment.…”