How internal cavities destabilize a protein

Abstract

Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å^-3.

Keywords: high-pressure NMR; protein folding and cooperativity; protein stability; unfolded state.

Fig. 1.

Pressure-dependent protein stability from NMR measurements. (A) Backbone ribbon representation of L99A T4L (PDB ID code 1L90). The gray space-filling shapes represent the hydrated, hydrophilic (1 and 2), and empty, hydrophobic cavities (3 and 4) in the structure. (B) Residue-specific stabilities determined as a function of pressure with native-state HX, shown for selected amide hydrogens (colored symbols). The lines represent fits to Eqs. 1 and 2 in the text.

Fig. 2.

Effect of mutating Leu99 to Ala on the local stability to unfolding ΔG° for T4L as determined by native-state HX. Black, WT* T4L (37); blue, L99A T4L.

Fig. 3.

Residue-specific pressure-dependent stability data for L99A T4L. (A) Relative stabilities ΔG° for the native, partially and fully unfolded conformations of L99A T4L identified in this study. To the sides, protein structures are shown for a hierarchic model of protein unfolding. (B) Correlation of residue-specific ΔG° and ΔV° defines a broad range of stabilities for the CTD from which the cost of cavity formation was determined from a linear fit through the data.

Fig. 4.

Structure relaxation of L99A followed by relaxation dispersion NMR spectroscopy. (A) Decrease of ΔG_GE with pressure indicates that E becomes increasingly populated; (B) forward (k_GE) and reverse (k_EG) rate constants with pressure show that the exchange rate k_ex = k_GE + k_EG decreases with pressure. Global fitting yields activation volumes ΔV_GE^0‡ = −28 ± 2 mL/mol, ΔV_EG^0‡ = 9 ± 1 mL/mol, and ΔV_GE⁰ = −37 ± 2 mL/mol.