The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain

Abstract

Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35-residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic-aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D-NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair-wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic-aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.

Fig. 1.

The aromatic cluster in the VHP subdomain. The side chains of phenylalanines 47, 51, and 58 are shown in space filling representation in gray. Other side chains are shown as sticks. Other side chains in the hydrophobic core are in gray. The backbone is shown as a ribbon. This figure was produced using MOLMOL (Koradi et al. 1996), from the Protein Data Bank file 1VII.PDB.

Fig. 2.

Far-UV CD spectra of VHP subdomain mutants at 4°C. (a) Single leucine point mutants. (b) Double leucine mutants. Samples were 40 to 60 μM protein, in 50 mM phosphate buffer, pH 7.0. The M53L spectrum is shown in both (a) and (b) for comparison. HP36 (+); M53L (filled diamonds); F47L (open circles); F51L (open squares); F58L (open triangles); F47,51L (filled triangles); F47,58L (filled squares); F51,58L (filled circles).

Fig. 3.

Near-UV CD spectra of the VHP subdomain mutants at 4°C. (a) Single leucine point mutants. (b) Double leucine mutants. Samples were 100 to 225 μM protein, in 50 mM phosphate buffer, pH 7.0. The M53L spectrum is shown in both (a) and (b) for comparison. HP36 (+); M53L (filled diamonds); F47L (open circles); F51L (open squares); F58L (open triangles); F47,51L (filled triangles); F47,58L (filled squares); F51,58L (filled circles).

Fig. 4.

Thermal unfolding of the VHP subdomain mutants monitored by CD at 222 nm. (a) Single mutants. (b) Double mutants. Samples were 40 to 60 μM protein in 50 mM phosphate buffer, pH 7.0. For clarity, only every fourth data point is shown. For reference, the thermal unfolding of M53L is repeated in plot (b). HP36 (+); M53L (filled diamonds); F47L (open circles); F51L (open squares); F58L (open triangles); F47,51L (filled triangles); F47,58L (filled squares); F51,58L (filled circles).

Fig. 5.

Gel filtration chromatography of the VHP subdomain single (a) and double (b) mutants. The column was run at 4°C at 0.5 mL/min in 10 mM phosphate buffer pH 7.0 with 150 mM NaCl. Traces corresponding to each peptide are labeled on the figure. The gray traces are molecular weight standards that were spiked with HP36 in (a) only. Note that the chromatograms in (a) and (b) were run on different, but identical HiPrep 16/60 Sephacryl S-100 columns (Pharmacia Biotech) so the elution volumes of both the peptides and molecular weight markers vary slightly between the two figures.

Fig. 6.

Downfield region of the 1D-NMR spectra of VHP subdomains at 4°C. NMR samples contained approximately 1 mM protein, in 10 mM phosphate buffer pH 7.0.