Effects of temperature on the fluorescence intensity and anisotropy decays of staphylococcal nuclease and the less stable nuclease-conA-SG28 mutant

Abstract

Frequency-domain fluorescence spectroscopy was used to investigate the effects of temperature on the intensity and anisotropy decays of the single tryptophan residues of Staphylococcal nuclease A and its nuclease-conA-SG28 mutant. This mutant has the beta-turn forming hexapeptide, Ser-Gly-Asn-Gly-Ser-Pro, substituted for the pentapeptide Tyr-Lys-Gly-Gln-Pro at positions 27-31. The intensity decays were analyzed in terms of a sum of exponentials and with Lorentzian distributions of decay times. The anisotropy decays were analyzed in terms of a sum of exponentials. Both the intensity and anisotropy decay parameters strongly depend on temperature near the thermal transitions of the proteins. Significant differences in the temperature stability of Staphylococcal nuclease and the mutant exist; these proteins show characteristic thermal transition temperatures (Tm) of 51 and 30 degrees C, respectively, at pH 7. The temperature dependence of the intensity decay data are shown to be consistent with a two-state unfolding model. For both proteins, the longer rotational correlation time, due to overall rotational diffusion, decreases dramatically at the transition temperature, and the amplitude of the shorter correlation time increases, indicating increased segmental motions of the single tryptophan residue. The mutant protein appears to have a slightly larger overall rotational correlation time and to show slightly more segmental motion of its Trp than is the case for the wild-type protein.

FIGURE 1:

Phase and modulation data for the intensity decay of wild-type nuclease. The solid lines show the best single-exponential fit.

FIGURE 2:

Phase and modulation data for the intensity decay of mutant nuclease-conA-SG28. The solid lines show the best single-exponential fit.

FIGURE 3:

Temperature dependence of the preexponential α_i and τ_i for biexponential fits for wild-type (top) and mutant (bottom) proteins. Long lifetime parameters (●) and short lifetime parameters (■) are given.

FIGURE 4:

Temperature dependence of the preexponential α_i and τ_i for triexponential fits for wild-type (top) and mutant (bottom) proteins. Long lifetime (●), medium lifetime (▲), and short lifetime (■) are given. In the lifetime profiles (right), the dashed lines are the temperature-dependence profiles from the global Arrhenius analysis (see Discussion). For the wild-type, these dashed lines are for E_a,1 = 1.0 kcal/mol and A₁ = 0.987 ns⁻¹ for τ₁; E_a,2 = 1.76 kcal/mol and A₂ = 8.54 ns⁻¹ for τ₂; and E_a,3 = 4.0 kcal/mol and A₃ = 1901 ns⁻¹ for τ₃.

FIGURE 5:

Lifetime distribution fit for the tryptophan intensity decay of the nuclease-conA-SG28 mutant staphylococcal nuclease. The values of χ_R² are 81.7 and 0.8 for the unimodal and bimodal fits, respectively.

FIGURE 6:

Differential phase and modulated anisotropy data for anisotropy decays of wild-type nuclease. The solid lines show the best fit to two correlation times.

FIGURE 7:

Differential phase and modulated anisotropy data for anisotropy decays of nuclease-conA-SG28. The solid lines show the best fit to two correlation times.

FIGURE 8:

Amplitudes of the anisotropy decay for wild-type nuclease (top) and mutant nuclease-conA-SG28 (bottom) as a function of temperature.

FIGURE 9:

Temperature dependence of the overall (θ₁) correlation times for wild-type nuclease (○) and mutant nuclease-conA-SG28 (●). The dashed line shows the temperature dependence of η/T.

FIGURE 10:

Temperature dependence of the α₁(○)and α₃ (□)for the global analysis or data sets with linkage via the Arrhenius equation (see Discussion). Values for α₂ are not shown, but they are 1 – α₁ – α₃ and generally show a temperature dependence similar to that for α₃ (but with an inflection at lower temperature, especially for the wild-type). The dashed line through these α₁ and α₃ points are fits of eq 6 for an assumed two-state transition. For the wild-type, this fit is for ΔH^o_UN = 55.9 kcal/mol, T_m = 48.0 °C, α_N = 0.885, and α_U = 0.019 for α₁ and ΔH^o_UN = 79.9, T_m = 53.1 °C, α_N = 0.091, and α_U = 0.615 for α₃. For the mutant, this fit is for ΔH^o_UN = 48.9 kcal/mol, T_m = 32.2 °C, α_N = 0.746, and α_U = −0.022 for α₁, and ΔH^o_UN = 41.8 kcal/mol, T_m = 34.6 °C, α_N = 0.152, and α_U 0.555 for α₃.