An influence of positive charge distribution in the peptide sequence?

The addition of GAGs accelerated aggregation kinetics of all three peptides studied, however to a different extent. The difference observed in aggregation kinetics among three peptides could be due to the distribution of positive charges within the peptide sequence. All three peptides and their charge distribution along the amino acid sequence are shown in table 4.7. Both somatostatin (K4 and K9) and LHRH (H2 and R8) have two positively charged amino acids that are located distantly in their sequence. On the other hand, substance P has two positively charged amino acids (R1 and K3) that localized towards the N-terminal of the sequence

127 Table 4.7: Amino acid sequence of neuropeptides studied and their charge distribution.

According to the previously published reports GAGs-peptide interactions occurs via binding of negatively charged sulphate groups of GAGs to positively charged amino acids in the sequence. In the case of substance P, since all positive charges are localized towards one end of the sequence, the rest of the sequence might not be actively interacting with heparin. Overall interaction of substance P with heparin hence might be poor. In contrast, previous reports have proposed that availability of a high number of basic residues and presence of basis-nonbasic- basic (B-N-B) motif in the peptide/protein sequence facilitates strong peptide/protein-heparin interactions [9, 36, 37]. We argue that even though, substance P has a B-N-B motif (R1-P2- K3) in their sequence, substance P contains fewer basic residues (2 basic residues) and more importantly, these two basic residues are localized towards one terminal resulting poor interactions of heparin with rest of the sequence. Other plausible explanation for the case of substance P could be that substance P converts to thermodynamically stable confirmation prior to the binding of GAG molecules leading to overall fewer interactions with heparin. In other words, during the self-assembly process substance P discover their thermodynamically stable conformation more promptly than somatostatin and LHRH without leaving time for heparin to bind. This assumption is supported by slower aggregation kinetics observed for somatostatin and LHRH and rapid aggregation kinetics observed for substance P in the absence of heparin. In the case of somatostatin and LHRH, heparin has sufficient time to bind to the peptide as they require more time to find their thermodynamically stable conformation. Since substance P has poor interactions with heparin, the molecular level arrangement of both substance P-heparin and pure substance P assemblies are similar as evidenced by the presence of parallel β-sheet

128 network detected by IR spectroscopy and similar form factor observed for nanotubes formed both in the presence and absence of heparin by SAXS. SAXS data also shows that heparin has no influence on the structure of substance P nanotubes as supported by similar diameter and wall thickness for both heparin-mediated nanotubes and pure substance P nanotubes. This finding supports our hypothesis that heparin has poor interactions with substance P and these interactions begin after substance P finding their thermodynamically stable conformation. In other words, heparin interacts with substance P towards the end of the self-assembly process.

4.5 Conclusion

In this work, we studied and compared the detailed mechanism of aggregation and amyloid formation by somatostatin both in the presence and absence of GAGs. In addition, self- assembly of LHRH and substance P both in the presence and absence of GAGs were also examined and compared to a lesser extent. Our biophysical data suggest that GAGs possess a distinct effect on self-assembly of somatostatin, LHRH and substance P which we assume is due to the differences in positive charge distribution along their amino acid sequence. More importantly, our results indicate that heparin modifies not only aggregation kinetics but also the structure of the assemblies formed. A similar type of results has been reported lately by others. These data further confirm our postulate that heparin acts not as just an inert aggregation inducer, but significantly changes the molecular structure of most amyloid-forming systems. It is hence important to carefully consider the structural effects of GAGs on peptide/protein self- assemblies when investigating amyloids and using such aggregation helpers, especially when concluding on structure-function relationships or when investigating amyloid-based nanostructures as bionanomaterials.

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CHAPTER 5