Probing the Formation of Stable Tertiary Structure in a Model Miniprotein at Atomic Resolution:  Determinants of Stability of a Helical Hairpin

Abstract

The minimal model system to study the basic principles of protein folding is the hairpin. The formation of beta-hairpins, which are the basic components of antiparallel beta-sheets, has been studied extensively in the past decade, but much less is known about helical hairpins. Here, we probe hairpin formation between a polyproline type-II helix and an alpha-helix as present in the natural miniprotein peptide YY (PYY). Both turn sequence and interactions of aromatic side chains from the C-terminal alpha-helix with the pockets formed by N-terminal Pro residues are shown by site-directed mutagenesis and solution NMR spectroscopy in different solvent systems to be important determinants of backbone dynamics and hairpin stability, suggesting a close analogy with some beta-hairpin structures. It is shown that multiple relatively weak contacts between the helices are necessary for the formation of the helical hairpin studied here, whereas the type-I beta-turn acts like a hinge, which through certain single amino acid substitutions is destabilized such that hairpin formation is completely abolished. Denaturation and renaturation of tertiary structure by temperature or cosolvents were probed by measuring changes of chemical shifts. Folding of PYY is both reversible and cooperative as inferred from the sigmoidal denaturation curves displayed by residues at the interface of the helical hairpin. Such miniproteins thus feature an important hallmark of globular proteins and should provide a convenient system to study basic aspects of helical hairpin folding that are complementary to those derived from studies of beta-hairpins.

Infrared Study of the Folding Mechanism of a Helical Hairpin: Porcine PYY

“…To better quantify the folding/unfolding thermodynamics of PYY and the mutants studied here (see below), we globally analyzed their CD melting curves measured at 222 nm using a global fitting method (see Methods Section for detail). As shown (Figure 3 and Table 1), the thermal melting temperature ( T m ) of wild-type PYY thus obtained is 44.0 ± 1.6 °C, which is almost identical to that determined by Zerbe and coworkers using NMR spectroscopy (11). …”

“…However, kinetically this can be achieved by either increasing the folding rate or decreasing the unfolding rate. To dissect these possibilities, we studied the thermodynamic and kinetic effects of two mutations (i.e., Y21A and Y27A) that weaken the hydrophobic interactions between the two helices (11). As expected, both mutations destabilize the PP-fold (Figure 3 and Table 1), although the Y27A mutation has a much larger effect than the Y21A mutation, which is in agreement with the NMR measurements of Zerbe and co-workers (11).…”

“…In addition, we investigated the folding kinetics of the A7Y mutant, which was previously shown to be more stable than the wild-type peptide (11). Our CD results confirm that the A7Y mutation increases the thermal stability of the PP fold (Figure 3 and Table 1).…”

“…As shown (Figure 1), porcine PYY (referred to hereafter as PYY) is free of disulfide bonds and assumes a structure known as the PP-fold in which an N-terminal type II polyproline (PPII) helix folds onto a C-terminal α-helix to form a stable, well defined hydrophobic cluster between the interfaces of the two helices (11, 16, 17). To dissect the kinetic roles of the two most important structural elements in the PP-fold, i.e., the hydrophobic cluster and the turn, we studied the folding-unfolding kinetics of two sets of PYY mutants.…”

Non-specific binding and general cross-reactivity of Y receptor agonists are correlated and should importantly depend on their acidic sectors

“…Residues 4–25 are not critical for binding to the Y2 receptor (see [45]). Residues in the helical 18–32 sector of all primary Y agonists (see pdb files 1RU5, 1RUU [27], 2DEZ [35] and 2RK [34] for PYY, 1F8P [2] and 1FVN [3] for NPY, 1BBA [29] and 1LJV [26] for bPP), and especially in the 19–23 zone (pdb files 1RU5 and 1RUU [27]) are known to be important in subtype selectivity of Y agonists. The C-terminal hexapeptide, while critical for the specific attachment of agonists to all Y receptors, may not be important in the non-specific binding, since this binding does not differ between Y1 or Y2 -selective NPY and PYY analogs and the respective parent peptides (Fig.…”