Intermolecular interactions are of fundamental importance to fully comprehend a wide range of protein behaviors such as oligomerization, folding and recognition. Two peptides, NPY([18-36]) and NPY([15-29]), segmented from human neuropeptide Y (hNPY), were synthesized in this work to study the interaction between species. Information about intermolecular interactions was extracted from their oligomerizing behaviors. The results from CD and NMR showed that the addition of 2, 2, 2-trifluoroethanol (TFE) induces a stable helix in each peptides and an extended helix in NPY([18-36]), formed between residues 30-36. Pulsed field gradient NMR data revealed that NPY([15-29]) forms a larger oligomer at lower temperatures and continuously dissociates into the monomeric form with increasing temperature. NPY([18-36]) was also found to undergo an enhanced interaction with TFE and a more favorable self-association at higher temperatures. We characterized the changes of oligomerized states with respect to temperature to infer the effects of entropy and interaction energy on the association-dissociation equilibrium. As shown by NPY([15-29]), deletion of helical secondary structure or residues from the C-terminal segment may disrupt the solvation by TFE and results in entropy increase as the oligomer dissociates. Unlike that in NPY([15-29]), the extended helix in NPY([18-36]) improves the binding of TFE, and as a result, entropy is gained via the transfer of the TFE cluster from the interface between monomeric peptides into the bulk solvent. This observation suggests that the oligomerized state may be modulated by the entropy and energetics contributed by helical segments in the oligomerization process.
Understanding the complex relationship between amino acid sequence and protein behaviors, such as folding and self-association, is a major goal of protein research. In the present work, we examined the effects of deleting a C-terminal residue on the intrinsic properties of an amphapathic α-helix of mastoparan-B (MP-B), an antimicrobial peptide with the sequence LKLKSIVSWAKKVL-NH2. We used circular dichroism and nuclear magnetic resonance to demonstrate that the peptide MP-B([1-13]) displayed significant unwinding at the N-terminal helix compared with the parent peptide of MP-B, as the temperature increased when the residue at position 14 was deleted. Pulsed-field gradient nuclear magnetic resonance data revealed that MP-B forms a larger diffusion unit than MP-B([1-13]) at all experimental temperatures and continuously dissociates as the temperature increases. In contrast, the size of the diffusion unit of MP-B([1-13]) is almost independent of temperature. These findings suggest that deleting the flexible, hydrophobic amino acid from the C-terminus of MP-B is sufficient to change the intrinsic helical thermal stability and self-association. This effect is most likely because of the modulation of enthalpic interactions and conformational freedom that are specified by this residue. Our results implicate terminal residues in the biological function of an antimicrobial peptide.
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