Structural characterization of a mutant peptide derived from ubiquitin: Implications for protein folding

Abstract

The formation of the N-terminal b-hairpin of ubiquitin is thought to be an early event in the folding of this small protein.Previously, we have shown that a peptide corresponding to residues 1-17 of ubiquitin folds autonomously and is likely to have a native-like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U~1-17!T9D, the native conformation is stabilized with respect to the wild-type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U~1-17!T9D peptide does indeed form a hairpin with a native-like register and a type I turn with a G1 b-bulge, as in the full-length protein. The reason for the greater stability of the U~1-17!T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys11, which may indicate that a charge-charge interaction between these residues is responsible.Keywords: b-hairpin; b-sheet; NMR; peptide; peptide conformations; structure; ubiquitin; X-PLOR The b-hairpin is one of the most commonly occurring structural motifs in globular protein folds: most antiparallel b-sheets contain at least some strand pairs that are contiguous in sequence and connected by a relatively tight turn. There has also been much speculation and some experimental evidence to support the idea that hairpins may play a key role in initiating the assembly of b-structures in folding~Ptitsyn, 1991; Muñoz et al., 1997!. In recent years, there has been considerable interest in using b-hairpins as model systems for developing our understanding of b-structure and several examples both of protein fragments and of de novo designed peptides capable of folding autonomously as hairpins have now been identified~Blanco et al., 1994;Searle et al., 1995;Ramirez-Alvarado et al., 1996!. The lessons learned from exploring the effects of sequence variations in these systems have been reviewed recently~Griffiths- Jones et al., 1998;Ramirez-Alvarado et al., 1999!. Sequence variations cannot only alter the stability of hairpin structures, but also the register of the b-strands. In the arms of the hairpin, a high b-propensity of individual residues is important for stability. However, specific pairwise contacts, especially between side chains interacting across the hairpin, are also important and optimization of these is hypothesized to be an important contribution in fixing the strand register~Smith & Regan, 1995;Wouters & Curmi, 1995;Hutchinson et al., 1998!. The turn sequence is also important: it is clear from analysis of hairpins within intact protein structures that some turn types are prevalent in b-hairpins. Sequence compatibility with these preferred structures has been shown in peptide studies to be an important determinant of structural specificity and stability~Ramirez- Alvarado et al., 1997;De Alba et al., 1999;Griffiths-Jones et al., 19...

Tryptophan zippers: Stable, monomeric β-hairpins

“…The estimated population of the folded hairpin was only 64%; nevertheless, the NMR resonances were unusually well resolved, and it was possible to attribute particular NOEs to the unfolded state. This deconvolution of the NOE data allowed the calculation of folded-state structures with good geometry and no distance restraint violations Ͼ 0.5 Å (27). The precision of the ensemble (backbone rmsd ϭ 0.59 Å) is close to that of the trpzips (see Table 3); however, as those authors note, the structure of the folded state of U(1-17)T9D may be more dynamic than indicated by the final ensemble (27).…”

“…3A). Cross-strand tryptophan † Very recently, the structure of U(1-17)T9D, a variant of the N-terminal ␤-hairpin of ubiquitin, was reported (Protein Data Bank code 1E0Q) (27). The estimated population of the folded hairpin was only 64%; nevertheless, the NMR resonances were unusually well resolved, and it was possible to attribute particular NOEs to the unfolded state.…”

“…This deconvolution of the NOE data allowed the calculation of folded-state structures with good geometry and no distance restraint violations Ͼ 0.5 Å (27). The precision of the ensemble (backbone rmsd ϭ 0.59 Å) is close to that of the trpzips (see Table 3); however, as those authors note, the structure of the folded state of U(1-17)T9D may be more dynamic than indicated by the final ensemble (27). Thermal melts were acquired with 20 M peptide samples in 20 mM potassium phosphate, pH 7.0. ϵ reduced apparent molecular weight, as determined from sedimentation data fit to a nonideal single-species model (see Materials and Methods).…”

Structure–function–folding relationship in a WW domain

“…Ala, Asp, and Gly occupy the (i ϩ 1) to (i ϩ 3) positions, respectively. Asp exhibits the highest statistical occurrence at the (i ϩ 2) position (30)(31)(32), whereas Gly is suited best to adopt the positive ͞-dihedral angles at the bulge position (i ϩ 3) in the type-I G1 bulge turn geometry (28,(32)(33)(34). The sequences corresponding to variants 3 and 4 were designed to test whether repositioning the functional Arg residue at the (i ϩ 1) or (i ϩ 2) position in a shorter sequence expected to adopt a type-I G1 bulge loop would yield WW domains with ligand-binding affinities comparable with variant 1.…”

Measuring the refolding of β-sheets with different turn sequences on a nanosecond time scale

“…Several peptide models with a basic hairpin structure have been used to examine the relationship between turn sequence and turn conformation. They are either designed peptides (8)(9)(10)(11)(12)(13) or short peptide segments adopted from protein sequences like ubiquitin (14)(15)(16)(17) and the protein G B1 domain (18,19). From these studies, there is little doubt that turn residues determine the conformation and stability of ␤-hairpins.…”