Abstract
Because of their limited size and complexity, de novo designed proteins are particularly useful for the detailed investigation of folding thermodynamics and mechanisms. Here, we describe how subtle changes in the hydrophobic core of a model three-helix bundle protein (GM-0) alter its folding energetics. To explore the folding tolerance of GM-0 toward amino acid sequence variability, two mutant proteins (GM-1 and GM-2) were generated. In the mutants, cavities were created in the hydrophobic core of the protein by either singly (GM-1; L35A variant) or doubly (GM-2; L35A/I39A variant) replacing large hydrophobic side chains by smaller Ala residues. The folding of GM-0 is characterized by two partially folded intermediate states exhibiting characteristics of molten globules, as evidenced by pressure-unfolding and pressure-assisted cold denaturation experiments. In contrast, the folding energetics of both mutants, GM-1 and GM-2, exhibit only one folding intermediate. Our results support the view that simple but biologically important folding motifs such as the three-helix bundle can reveal complex folding plasticity, and they point to the role of hydrophobic packing as a determinant of the overall stability and folding thermodynamic of the helix bundle.
Highlights
Proteins that explore multiple regions of the free energy surface by redesign represent versatile tools for the elucidation of folding mechanisms
The present findings indicate that the helix bundle GM-0 populates two equilibrium intermediate states, whereas only one folding intermediate is populated in both hydrophobic core mutants
Replacement of Leu-35 (GM-1 variant) and Leu-35/Ile-39 (GM-2 variant) by smaller side-chain alanine residues resulted in major decreases in the stabilities of the native-like states of the variants compared with GM-0 (Fig. 1)
Summary
All other reagents were of the highest analytical grade available. Protein Synthesis—The design and synthesis of GM-0 was based on that of the previously described de novo designed protein ␣3-1 [8], with two amino acid replacements at positions 42 (leucine to methionine) and 51 (cysteine to valine). The N terminus of the protein is acetylated, and the C terminus is amidated to avoid charge repulsion. GdnHCl Denaturation—GM-0, GM-1, and GM-2 (5 M for all proteins) were incubated for 2 h at 23 °C in 20 mM Tris-HCl, 130 mM NaCl, pH 7, in the presence of increasing concentrations of GdnHCl. Control.
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