On the unyielding hydrophobic core of villin headpiece

Abstract

Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solventexposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (a-helix to p-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.

Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure

“…To investigate the utility of the Trp-M Se pair as a probe of local α-helical structure in globular proteins, a mutant of the 36-residue villin headpiece helical subdomain (HP36) was prepared. HP36 is a small, autonomously folding three-helix protein that has been used widely as a model system for both computational and experimental studies of protein folding. ,− HP36 possesses a naturally occurring Trp residue at position 24. We mutated Asn 28 to M Se to create an i , i + 4 arrangement that places the side chains roughly one turn apart, bringing them into close contact in the helical state.…”

Thermodynamic origin of α-helix stabilization by side-chain cross-links in a small protein

“…32,33 VHP is a widely used model system in the study of folding kinetics and thermodynamics in short, fast-folding sequences. [34][35][36][37][38][39][40] To avoid complications from methionine oxidation during synthesis and biophysical analysis, we replaced the two Met residues in VHP with hydrocarbon isostere norleucine to generate sequence 1 (Fig. 1).…”

Selenomethionine, p-cyanophenylalanine pairs provide a convenient, sensitive, non-perturbing fluorescent probe of local helical structure

“…20 The hydrophobic core of HP36 contains three closely packed Phe residues, leading to characteristic ring current shifted resonances in the 1 H-NMR spectrum which is indicative of the folded state. 17,27,28 These peaks are observed in the 1 H-NMR spectrum of the F CN -M Se variant, providing additional evidence that the substitutions do not perturb the fold (Fig. S3, ESI †).…”

“…The domain has been widely used for studies of protein folding, dynamics and stability. [18][19][20][21][22][23][24][25][26][27] We replaced Trp-24 with F CN and residue 28 with M Se . These sites are located on the surface of the protein on the C-terminal helix.…”