| Summary: | 博士 === 國立臺灣大學 === 生化科學研究所 === 90 === Proteins possess the ultimate roles of executing biological processes, and more research efforts are directed toward functional and structural analysis of these macromolecules. Among a myriad of laboratory techniques, biophysical methods are mainly used for dissecting the properties and structures of proteins. From the structural information, structural-functional relationship can then be established. Studies on three different types of macromolecules, fusion peptide, snake toxin, and cytotoxic ribonucleases, using combinations of various fundamental or advanced biophysical techniques, have been carried out. The results are presented in this thesis.
Part I
Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point-mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu2-Glu11 and Trp14-Ile18, form amphipathic helical conformations, with Gly12-Gly13 forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp14-Ile18 reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion-peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.
Part II
Bungarus fasciatus IX (BF9), a chymotrypsin inhibitor, consists of 65 amino acid residues with three disulfide bridges. It was isolated from the snake venom of Bungarus fasciatus by ion exchange chromatography and belongs to the bovine pancreatic trypsin inhibitor (BPTI)-like superfamily. It showed a dissociation constant of 5.8 x 10-8 M with a-chymotrypsin as measured by a Langmuir binding isotherm using BIAcore binding assay system. The isothermal titration calorimetry revealed a 1:1 binding stoichiometry between this inhibitor and chymotrypsin and apparently no binding with trypsin. We have further used CD and NMR to determine the solution structure of this venom-derived chymotrypsin inhibitor. The 3D NMR solution structures of BF9 were determined on the basis of 582 restraints by simulated annealing and energy minimization calculation. The final set of 10 NMR structures was exceptionally well defined with average RMSD of 0.47 A for the backbone atoms in the secondary structure regions and 0.86 A in residues 3-58. Based on the 3D structure, we identified that the unusual chemical shifts observed for BF9 resulted from an aromatic ring current effect. The side chains of Phe23, Ty24, Tyr25, Phe35 and Phe47 exhibited many long-range NOEs and were the principal components of the hydrophobic core in BF9. To gain insight into the structure-function relationships among the proteins in the BPTI-like superfamily, we compared the 3D structure of BF9 with three BPTI-like proteins that possess distinct biological functions. We found that these proteins possessed similar secondary structure elements, but the loop regions and b-turn were different from one another. Based on residues at the functional site of each protein, we suggested that the flexibility, the rigidity, and the variations of the amino acid residues in both the loop and the b-turn regions are related to their biological functions.
Part III
Cytotoxic ribonucleases, isolated from oocyte and liver of bullfrog Rana catesbeiana, possess different base specificities, ribonucleolytic activities and cytotoxicities. To gain insight into the structure/function relationships on these ribonucleases, we cloned, expressed and purified recombinant as well as mutant proteins for several biophysical studies. CD experiments are used to check conformational stability, secondary structure and structural change at different pH values. Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) techniques are used for binding studies. NMR studies, including structure determination, protein folding, and dynamics, are carried out using native and 15N and/or 13C-labeled proteins. The following results will be presented.
(1) Three dimensional solution structures of recombinant RC-RNase 2 and RC-RNase 4 have been determined.
(2) Based on the biophysical data, we concluded that the reduction in catalytic and cytotoxic activities for the recombinant protein, which contains an extra Met residue and has a Gln instead of pyroglutamate at the N-terminus, are mainly due to the loss of two H-bonds in the N-terminal Gln residue.
(3) Using RC-RNase 4''s mutant proteins, we confirmed that Trp15 plays an important role in its high thermostability and causes a unique characteristic of CD data with an additional ellipticity minimum at 228 nm.
(4) The substrate-related residues in the base specificity of CpG vs. UpG among RC-RNase, RC-RNase 2 and RC-RNase 4 are derived using the NMR chemical shift mapping between free- and complex- structures. In addition, the binding characteristic based on SPR and ITC experiments were performed.
(5) The complex model of ribonuclease inhibitor (RI) and RC-RNases model revealed that RC-RNases evade RI and catalyze cleavage of cellular RNA, which leads to cell death.
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