Reinvestigation of the proposed folding and self‐association of the Neuropeptide Head Activator

Abstract

The Neuropeptide Head Activator (HA), pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe (pGlu is pyroglutamic acid), is involved in head-specific growth and differentiation processes in the freshwater coelenterate Hydra attenuata. Peptides of identical sequence have also been isolated from higher-organism tissues such as human and bovine hypothalamus. Early studies by molecular sieve chromatography suggested that HA dimerizes with high affinity (K d ≈ 1 nM). This dimerization was proposed to occur via antiparallel ␤-sheet formation between the Lys 7 -Phe 11 segments in each HA molecule. We conducted biophysical studies on synthetic HA in order to gain insight into its structure and aggregation tendencies. We found by analytical ultracentrifugation that HA is monomeric at low millimolar concentrations. Studies by 1 H-NMR revealed that HA did not adopt any significant secondary structure in solution. We found no NOEs that would support the proposed dimer structure. We probed the propensity of the Lys 7 -Phe 11 fragment to form antiparallel ␤-sheet by designing peptides in which two such fragments are joined by a two-residue linker. These peptides were intended to form stable ␤-hairpin structures with cross-strand interactions that mimic those of the proposed HA dimer interface. We found that the HA-derived fragments may be induced to form intramolecular ␤-sheet, albeit only weakly, when linked by the highly ␤-hairpin-promoting D-Pro-Gly turn, but not when linked by the more flexible Gly-Gly unit. These findings suggest that the postulated mode of HA dimerization and the proposed propensity of the molecule to form discrete aggregates with high affinity are incorrect.

Gas phase dimerization of neuropeptide head activator analogs useful for the noncovalent constraint of peptides

“…The results thus give no evidence of solution peptide dimerization, but these two peptides do appear to constrain the Ci2b 18mer in solution when fused to its N‐ and C‐termini, giving T m s of 54.210 and 44.1°C, respectively. These results on solution dimer formation are similar to those of Lai and Gellman,9 who failed to find evidence for solution dimers of the neuropeptide head activator.…”

“…The half‐saturation concentration for dimer formation of SKVILFE of 12 μ M , when detected in the gas phase, suggests much weaker dimer formation than published earlier for similar peptides 8. Given the short peptide sequence, lack of sequence specificity for dimer formation, limited surface area,9 and loss of conformational entropy upon dimerization, this is not surprising. We have not observed significant amounts of trimers or tetramers of EFLIVKS, or of other analogs, by mass spectrometry.…”

“…Examples of peptides dimerizing in solution include amyloid Aβ (1–40),5 the relatively short peptide neuropeptide Y,6 gramicidin,7 and the peptide hormone neuropeptide head activator. This peptide (pyroglu‐PPGGSKVILF) has been reported to be inactivated by dimerization at levels below 10 −10 M ,8 although Lai and Gellman,9 using analytical ultracentrifugation and NMR, found no evidence for dimerization at millimolar peptide levels.…”

Enhanced therapeutic window for antimicrobial Pept-ins by investigating their structure-activity relationship

“…Next, we tested the impact of β-turn-promoting linkers on P2 antimicrobial potency, such as the reverse turn motif pG [39,40], which promotes β-hairpin formation and allows a more defined structure [41] by introducing a reverse turn [42]. The cyclic structure formed between the side chain and the N-H of proline (P) offers a turn and a structure rigidity, making proline ideal for a β-turn [43].…”

3-Azido-3-deoxy-2,2′:5,6-di-O-isopropylidene-2-C-hydroxmethyl-D-gulono-1,4-lactone

“…Gellman (Lai & Gellman, 2003;Hayen et al, 2004) and Seebach (Lelais & Seebach, 2003;Rueping et al, 2004) have pioneered studies on the exploitation of -amino acids in their adoption of secondary structural features in short chains. Although sugar amino acids (SAA) have been extensively investigated as peptidomimetics and dipeptide isosteres (Schweizer, 2002;Chakraborty et al, 2004), there have been very few studies of -SAAs Johnson et al, 2004), even though some oxetane-derived -SAAs exhibited novel helical structures (Barker et al, 2001).…”