NMR Based Structural Studies Of HaF Fusion Peptide And Potassium Channel Inhibitor ImKTx104

NMR (Nuclear Magnetic Resonance spectroscopy) is an essential tool in structural biology because it can give overall information of structure and dynamics of proteins in solution. During the past20years, the NMR method of resolving protein structures has made big progress to be more straightforward and automatic. Meanwhile, the development of NMR hardware instruments makes it possible to resolve protein structures of large molecular weight. In this paper, we studied the structures of two membrane function related peptides, mainly by solution NMR, showing the power of NMR in solving crucial biological problems.Topic One:The structures and fusion mechanism of the baculovirus fusion peptidesMembrane fusion is an essential step of viral infection. However, fusion mechanism has not been clearly understood yet. In this thesis, we solved the structures of wild-type HaF fusion peptide, wild-type LD130fusion peptide and four mutants of HaF fusion peptide (D11L、I2N、N1G、G3L) by NMR, and detected the locations of these peptides in SDS micelles by paramagnetic broadening experiments.The structure of the wild-type HaF fusion peptide is in an invert "V"(or boomerang) shape. Its4mutants, which have different fusion activities, show varied structures. We found that the rigid turn in the middle of this conformation and the complete amphiphilic surface are two essential factors of the fusion activity. On the other hand, wild-type LD130fusion peptide adopts a "pogo-stick" conformation, which contains amphiphilic surface but no kink inside the long linear helix, showing the diversity of the structures of fusion peptides. Based on the structures and locations of the fusion peptides in a simulated membrane environment, we infer the mechanism of inserting step during fusion process-the structure to baculovirus fusion peptide can be divided into two parts which are called "fusion region" and "structure region", respectively. And there is a synergic effect between the two parts to push the fusion peptide into membrane.Topic Two:Structural and dynamical studies of acidic scorpion potassium channel toxin ImKTx104Potassium channels have important effects on signal transmission in cells. Any mutation in potassium channels can lead to serious diseases. Scorpion venom peptides are nature and efficient potassium channel inhibitors, and they can specifically interact with the channels and cure relative diseases. Therefore, the structures of scorpion venom peptides can be used as templates of drug design. So it is of great interest to discover specific inhibitors from scorpion venom for various potassium channels.Recently, the first peptide inhibitor of KCNQ1channel-ImKTx104has been discovered. It is a new member of α-KTx subfamily, but its sequence has low homology to other toxins in this subfamily. We solved the structure and determined its dynamical characteristics by NMR experiments. The structure of ImKTx104mainly contains coiled-coil except a short310-helix. Dynamic studies and H-D experiments show that the structure is not flexible. Compared to other peptide in α-KTx subfamily, ImKTx104still maintains the properties of classical "CS-α/β fold" in space. Therefore, we think the structure of ImKTx104has a modified "CS-α/β fold", and the conserved disulfide bonds and "CXXXC---GXC---CXC" sequence is crucial to support the framework of this fold. The structure of ImKTx104expanded our knowledge of "CS-α/β fold", expressed the diversity of scorpion venom peptides, and also helped us to deeply explore the mechanism of the interaction between KCNQ1channel and its inhibitor.