High-pressure denaturation of apomyoglobin.
Academic Article
Overview
Research
Identity
Additional Document Info
Other
View All
Overview
abstract
The pressure denaturation of wild type and mutant apomyoglobin (apoMb) was investigated using a high-pressure, high-resolution nuclear magnetic resonance and high-pressure fluorescence techniques. Wild type apoMb is resistant to pressures up to 80 MPa, and denatures to a high-pressure intermediate, I(p), between 80 and 200 MPa. A further increase of pressure to 500 MPa results in denaturation of the intermediate. The two tryptophans, both in the A helix, remain sequestered from solvent in the high-pressure intermediate, which retains some native NOESY cross peaks in the AGH core as well as between F33 and F43. High-pressure fluorescence shows that the tryptophans remain inaccessible to solvent in the I(p) state. Thus the high-pressure intermediate has some structural properties in common with the apoMb I(2) acid intermediate. The resistance of the AGH core to pressures up to 200 MPa provides further evidence that the intrinsic stability of these alpha-helices is responsible for their presence in a number of equilibrium intermediates as well as in the earliest kinetic folding intermediate. Mutations in the AGH core designed to disrupt packing by burying a charge or increasing the size of a hydrophobic residue significantly perturbed the unfolding of native apoMb to the high-pressure intermediate. The F123W and S108L mutants both unfolded at lower pressures, while retaining some resistance to pressures below 50 MPa. The charge burial mutants, A130K and S108K, are not stable at very low pressures and both denature to the intermediate by 100 MPa, half of the pressure required for wild type apoMb. Thus a similar intermediate state is created independent of the method of perturbation, and mutations have similar effects on native state destabilization for both methods of denaturation. These data suggest that equilibrium intermediates that can be formed through different means are likely to resemble a kinetic intermediate.
