1H-NMR study of the effect of temperature through reversible unfolding on the heme pocket molecular structure and magnetic properties of aplysia limacina cyano-metmyoglobin

Abstract

Two-dimensional 1H NMR spectroscopy over a range of temperature through thermal unfolding has been applied to the low-spin, ferric cyanide complex of myoglobin from Aplysia limacina to search for intermediates in the unfolding and to characterize the effect of temperature on the magnetic properties and electronic structure of the heme iron. The observation of strictly linear behavior from 5 to 80 C degrees through the unfolding transition for all hyperfine-shifted resonances indicates the absence of significant populations of intermediate states to the cooperative unfolding with Tm approximately 80 degrees C. The magnetic anisotropies and orientation of the magnetic axes for the complete range of temperatures were also determined for the complex. The anisotropies have very similar magnitudes, and exhibit the expected characteristic temperature dependence, previously observed in the isoelectronic sperm whale myoglobin complex. In contrast to sperm whale Mb, where the orientation of the magnetic axis was completely temperature-independent, the tilt of the major magnetic axis, which correlates with the Fe-CN tilt, decreases at high temperature in Aplysia limacina Mb, indicating a molecular structure that is conserved with temperature, although more plastic than that of sperm whale Mb. The pattern of contact shifts reflects a conserved Fe-His(F8) bond and pi-spin delocalization into the heme, as expected for the orientation of the axial His imidazole.

FIGURE 1

Structure of the heme with the reference coordinate system x′, y′, z′ (R, θ′, Ω′) and magnetic coordinates system, x, y, z (R, θ, φ), shown. The x′, y′ plane is given by the mean plane of the heme in the crystal structure, with the x′ axis passing through the pyrrole B nitrogen, and the z′ axis normal to the heme and oriented to the proximal side. The positions relative to the heme of the residues whose proton dipolar shifts were used to generate the magnetic axes are also shown (proximal residues as rectangles, distal residues as circles). The angle α and κ = α + γ are defined; not shown is the angle β, which measures the tilt of the major magnetic axis, z, from the heme normal, z′.

FIGURE 2

Thermal denaturation of Aplysia metMbCN as a function of temperature in the range 20–92°C (293–365 K) in 100 mM phosphate pH 8.1 and 2 mM NaCN. The graph shows the CD data at 222 nm recorded at 1° intervals on a 4-μM protein solution (light path = 0.1 cm). The open triangles on the main graph are points recorded at single temperature values after equilibrating the protein solution for 10 min. The solid triangle indicates the value corresponding to a protein solution kept at 92°C for 10 min and slowly cooled down to 20°C. The continuous line is a fit to Eq. 4.

FIGURE 3

500 MHz ¹H-NMR spectra of Aplysia metMbCN as a function of temperature at pH 8.6. The previously assigned (12,30) resolved peaks are labeled for the major isomer (∼75%) in solution (M = methyl, P = propionate, and V = vinyl); minor component (∼25%) peaks are marked by small stars.

FIGURE 4

Plot of δ_DSS(obs) versus reciprocal absolute temperature (Curie plot) for the iron-ligated heme and axial His(F8) protons of Aplysia metmbCN in ²H₂O, 100 mM in phosphate, pH 8.6; 1-CH₃ (□), 3-CH₃ (▵), 5-CH₃ (▿), 8-CH₃ (○), α-meso-H (▪), β-meso-H (▴), γ-meso-H (▾), δ-meso-H (•), and His(F8) C_αH (★).

FIGURE 5

Plot of δ_DSS(obs) versus reciprocal absolute temperature (Curie plot) of nonligated Phe₉₈(FG2) in Aplysia metMbCN in ²H₂O; the solid circles, squares, and triangles represent C_δHs, C_ɛHs, and C_ζH shifts, respectively.

FIGURE 6

Plot of δ_dip(obs), obtained via Eq. 6 versus δ_dip(calc) obtained from the optimized magnetic axes (Eqs. 2 and 5) as a function of temperature, with the parameter for each temperature listed in Table 1, at (A) 7°C, (B) 25°C, (C) 47°C, and (D) 68°C. The 21 inputs include: F₄₃(CD1) C_αH; F₄₆(CD4) C_αH, C_δHs, C_ɛHs; I₆₇(E11) C_αH; A₉₂(F5) C_αH, C_βH₃; H₉₅(F8) C_αH; V₉₆(F9) C_βH, C_γ1H₃, C_γ2H₃; F₉₈(FG2) C_δHs, C_ɛHs, C_ζH; V₁₀₀(FG4) C_γ1H₃, C_γ2H₃; F₁₀₅(G5) C_ζH; and V₁₀₈(G8) C_αH, C_βH, C_γ1H, C_γ2H.

FIGURE 7

Plot of (A) angles, α, β, and κ, that define the orientation of the magnetic axes; and (B) the magnetic anisotropies determined from five-parameter least-square searches over the temperature range 7–68°C. The uncertainties in each parameter are given by vertical error bars.

FIGURE 8

Plot of δ_con versus reciprocal absolute temperature (Curie plot) for the heme methyls (1-CH₃, •; 3-CH₃, ○; 5-CH₃, ▾; and 8-CH₃, ▿).