NMR Based Structural Studies Of Several Peptides

Proteins are the carrier and functional executors of life. Studies on the function and structure relationship of protein is a key step to understand the secrets of life, which is also the basis of protein engineering and drug discovery. Nuclear magnetic resonance (NMR) spectroscopy is highly suitable to investigate the structures of peptide and protein in solution and can provide information about dynamics. We use solution NMR techniques to study influenza hemagglutinin (HA) fusion peptide and its two mutants, and calculate the structures of three conotoxin and a scorpion venom peptide. These structures facilate us to decipher the relationship between structure and function. The contents can be devided into two parts:1. NMR structure of fusion peptide of influenza hemagglutinin subtype3and its mutantsInfluenza virus remains a high priority for health concern. The key step of Influenza virus infection is the membrane fusion which delivers the genetic materials of virus into the cytoplasm of the infected host. HA is responsible for fusing process. Mutations in the HA allow virus to elude host immunity. The fusion peptide which is at the N-terminus of the HA is highly conserved which initiates the fusogenic process between viral and host cell membrane. Previous studies reported a boomerang shaped structure for a20-residue fusion peptide of H3-subtype (H3-HAfp20). In2010, Bax et al recognized that three conserved C-terminal residues Trp21-Tyr22-Gly23might have important impacts on the structure, which were not included in the previous studies. Hl-HAfp23shows a tight helical hairpin structure. There are two amino acids difference between HI-subtype and H3-subtype fusion peptide sequences and the Gly/Asn deviation of12th residue, which located at the turn region, is highly conserved. When the flexible Gly12at the turn region is substituted by Asn, it is not known whether the large sidechain of Asn12will hinder the formation of a hairpin structure. We expressed the13C,15N labelled fusion peptide of H3-subtype (named H3-HAfp23) in E.coli and determined its3D structure by heteronuclear NMR spectroscopy. The H3-HAfp23displays similar tight helical hairpin structure with the same helical and turn region as in H1-HAfp23. The difference is that the sidechain of Asn12points to the second helix and the stereo effect pushes the second helix Gly13-Tyr22towards the C-terminus. Therefore we can conclude that helix hairpin structure is conserved among16HA subtypes. In order to address the relationship between structure and function, we calculated the NMR structures of two mutants as well, a lethal G1V mutant and a G1S mutant which displays a hemifusion phenotype. G1S mutant adopts a helical hairpin structure the same as H3-HAfp23. However, inter-helical H-bond is only observed between the hydroxyl oxygen of Ser1sidechain and the NH on the indole ring of Trp21. The structure of G1V mutant is partially opened which is quite different from wild type H3-HAfp23. The N terminal and C-terminal region have no interaction. Compared with the wild type structure, we find that, with the loss of function, the hairpin structure is gradually opened, indicating the importance of hairpin structure. The charge-dipole interaction between N-and C-terminus is important for the penetration of the peptides into the DPC micelles which is also demonstrated by Mn2+titration.2. Structural studies of three conotoxin and a scorpion venom peptideLots of biological toxins are peptides which target at receptors/channels. Studies on the relationship of toxin structures and functions will deepen our understanding of molecular mechanism about the interaction between toxin and receptors/channels. Collaborating with other groups, we used2D proton homonuclear NMR methods to calculate the structures of three conotoxins and a scorpion venom peptide. All of the three conotoxins can relieve the pain. Eb1.6shows structure highly similar with other conotoxins from α-conotoxin4/7subtype. T1and L11are totally new structures. The structure and function relationship needs to be further investigated.Scorpion venom peptide BmP05can selectively block the small-conductance Ca2+-activated K+channel(SK channel) and its mutant BmP05-T can both block SK and voltage-gated potassium channel Kvl.3. Alanine screening experiments demonstrate that the β-sheet of BmP05-T interacts with Kv1.3and SK channel. The NMR structure of BmP05-T has a high similarity with ScyTx which shares a conserved amino acid sequence with BmP05-T, and the mutated amino acids change the charge distribution on the surface. Our results support the hypothesis that the acidic amino acids determine the binding interface of venom peptide and demonstrate that the β-sheet of BmP05-T interacts with SK channel, providing the structural information for further research.