Nascent Hairpins in Proteins: Identifying Turn Loci and Quantitating Turn Contributions to Hairpin Stability

Abstract

Many factors influence the stability of hairpins that could appear as foldons in partially folded states of proteins; of these, the propensity of certain amino acid sequences to favor conformations that serve to align potential β-strands for antiparallel association is likely the dominant feature. Quantitating turn propensities is viewed as the first step in developing an algorithm for locating nascent hairpins in protein sequences. Such nascent hairpins can serve to accelerate protein folding or, if they represent structural elements that differ from the final folded state, as kinetic traps. We have measured these "turn propensities" for the two most common turn types using a series of model peptide hairpins with four- and six-residue loops connecting the associated β-strands. Loops of four to six residues with specific turn sequences containing only natural l-amino acids and glycine can provide as much as 15 kJ/mol of hairpin stabilization versus loops lacking the defined turn loci. Single-site mutations within some of the optimal connecting loops can have ΔΔG effects as large as 9-10 kJ/mol on hairpin stability. In contrast to the near universal II'/I' turns of model hairpins, a number of hairpin-supporting XZZG sequence β-turns with α and/or γ configurations at the ZZ unit were found. A series of turn replacements (four-residue β-turns replaced by sequences that favor five- and six-residue reversing loops) using identical strands in our model systems have confirmed that several sequences have intrinsic turn propensities that could favor β-strand association in a non-native strand register and thus serve as kinetic traps. These studies also indicate that aryl residues immediately flanking a turn sequence can alter relative turn propensities by as much as 9-11 kJ/mol and will need to be a part of any nascent hairpin recognition algorithm.

Biological consequences of improving the structural stability of hairpins that have antimicrobial activity

“…Without the D‐Pro‐L‐Pro template to serve as the hairpin turn, we chose the Arg‐Arg residues in the middle of the sequence and replaced it with a D‐Arg‐L‐Arg sequence since a D‐amino acid followed by an L‐amino acid has been shown to promote the formation of a hairpin turn . These residues were chosen because they were present at the middle of the sequence and having a turn nucleating sequence in the middle of a loop has been shown to increase the hairpin fold stability of the adjacent residues .…”

“…In order to improve the stability of the peptide, we replaced the ‐GSG‐ loop with a hairpin turn, ‐NRRTGR‐. The new turn was chosen because Trp‐flanked [4:6] hairpin turns have been shown to be very stable and the residues N, T, and G at those specific positions have been shown to be crucial for enhanced hairpin stability . Due to the lack of specific residue preferences at the other positions, we chose to use Arg residues in order to increase the net positive charge and thus increase AMP selectivity.…”

Roles of Amphipathicity and Hydrophobicity in the Micelle‐Driven Structural Switch of a 14‐mer Peptide Core from a Choline‐Binding Repeat

“…This peptide system is probablyv ery sensitive to changes along the strands, because the sequence at the b-turn (Ile i Ala i + 1 Asp i + 2 Lys i + 3 ; Figure 2) is not optimal for b-hairpin stability, [19] and the turn is known to play an essential role in b-hairpin formation. [20] This intrinsic instability may contributet ot he ability of the peptide to switch between conformations when confrontedw ith changes in the environment. In this context of marginal stability,c ross-strand interactions may play an essential role in maintaining the hairpin architecture.…”

A pH Switch for β‐Sheet Protein Folding

“…This recently discovered pH switch was found during a mutational study on the site specific amino acid requirements of different types of turns in β-hairpins. In a reference β-hairpin system, NMR studies indicated that the mole fraction of the folded state went from χ F = 0.90 at pH 8 to < 0.1 at pH 2.5 [24] : ΔΔG F ≥ 11 kJ/mol.…”