Autonomous folding of a peptide corresponding to the N‐terminal β‐hairpin from ubiquitin

Abstract

The N-terminal 17 residues of ubiquitin have been shown by 1 H NMR to fold autonomously into a b-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly b-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for b-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the b-region of conformational space even in the absence of the hairpin structure.

Transient 2D IR spectroscopy of ubiquitin unfolding dynamics

“…Several experimental and simulation studies have reported sequential folding after the formation of a stable core that includes the strand I-II hairpin and the ␣-helix (36-40). The stability of this N-terminal 1-37 fragment has been investigated by fragmentation studies, multidimensional NMR experiments, and MD simulation (41)(42)(43)(44)(45). MD simulations suggested a transition state with ␤-strands I, II, and V intact and an unfolding pathway involving strands III-V before unfolding of the hydrophobic core (45,46).…”

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

“…This finding prompted the investigation of the potential for independent folding of peptide fragments derived from the N-terminus of ubiquitin. Later work showed that a peptide comprising the N-terminal 17 residues of the protein forms a native-like b-hairpin in both aqueous methanol and water, albeit at low apparent population~Cox et al, 1993;Zerella et al, 1999 Hairpin-forming peptides are proving valuable as model systems, but it is important to recognize that they may not be fully representative of b-structures in globular proteins: an isolated hairpin lacks the tertiary interactions that would help to define its geometry in a folded protein. An isolated hairpin may form nonnative intramolecular interactions that will compensate in part for the absence of tertiary interactions.…”

“…This finding prompted the investigation of the potential for independent folding of peptide fragments derived from the N-terminus of ubiquitin. Later work showed that a peptide comprising the N-terminal 17 residues of the protein forms a native-like b-hairpin in both aqueous methanol and water, albeit at low apparent population~Cox et al, 1993;Zerella et al, 1999!. Furthermore, protein engineering studies have re-vealed that residues 1-17 are involved in a productive intermediate populated at an early stage of the folding process~Khorasanizadeh et al, 1996!, suggesting that autonomous folding of this structural element might well play an important role in the folding mechanism of this protein.…”

Solution structure of a peptide model of a region important for the folding of ?-lactalbumin provides evidence for the formation of nonnative structure in the denatured state

“…Methods developed to predict the population of a conformer using Nuclear Overhauser Effect (NOE)/ROE intensities or 3 J NH␣ coupling constants often lead to contradictory or unsatisfactory results. 18,19 Such methods typically suffer from the inherent assumption of a two-state equilibrium between the structured conformer and the random coil state. In the peptide studied here, there appear to be at least two-folded conformations.…”