Energy landscape in protein folding and unfolding

Abstract

We use (1)H NMR to probe the energy landscape in the protein folding and unfolding process. Using the scheme ⇄ reversible unfolded (intermediate) → irreversible unfolded (denatured) state, we study the thermal denaturation of hydrated lysozyme that occurs when the temperature is increased. Using thermal cycles in the range 295 < T < 365 K and following different trajectories along the protein energy surface, we observe that the hydrophilic (the amide NH) and hydrophobic (methyl CH3 and methine CH) peptide groups evolve and exhibit different behaviors. We also discuss the role of water and hydrogen bonding in the protein configurational stability.

Keywords: energy landscape; hydration water; protein folding; proton NMR.

Fig. 1.

Protein ¹H NMR spectra (magnetization versus the chemical shift) of cycles A (Left) and E (Right). (Top) Spectra in the heating phase; (Bottom) spectra in the cooling phase. Cycle A regards the complete thermal denaturation of lysozyme, whereas cycle E deals with the reversible evolution from the native to the unfolded (intermediate) state. In both cycles the spectral evolution from the native to the denatured state is reported in different colors just to clarify the protein thermal behavior. The spectra of the native state are reported in green, those of the RU region before the onset of the $C_P\left(T\right)$ peak ($320-336$ K) in dark yellow, the spectra above this region up to the $C_P^{\text{max}}\left(T\right)$ ($337-347$ K) are in red, and finally spectra in the irreversible denatured region (IU) are in blue.

Fig. 2.

AR representation of the measured magnetization values of the hydrophilic amide groups (NH). Data for all five different studied thermal cycles (A, B, C, D, and E) are illustrated. The characteristic temperatures $T^{*}$ and $T_D$ are also reported. Lines represent AR behaviors; the corresponding activation energies $E_A$ are indicated in kcal/mol. Cycles A, B, and D deal with a complete denaturation; C operates in the native protein state (N), whereas E refers to the native and intermediate states (N$\rightleftarrows$ RU).

Fig. 3.

Thermal evolution of the magnetization, $M_I\left(T\right)$, of the protein methyl (${\text{CH}}_3$) groups for all of the studied thermal cycles. Lines and symbols are the same as used in Fig. 2.

Fig. 4.

Protein methine ($\text{CH}$) magnetization, $M_I\left(T\right)$, for all of the different thermal cycles. Lines and symbols are the same as previously used.