Structure And Mechanism Study Of Biological Function Of Antimicrobial Peptide Hedistin And Dihydrofolate Reductase Mutant DHFR-W21H

Nuclear magnetic resonance (NMR) spectroseopy is highly suitable to investigate the solution structure of peptide and protein. In this thesis, NMR spectroscopy was used to investigate the structure and mechanism of function of both antimicrobial peptide hedistin and dihydrofolate reductase mutant DHFR-W21H.1. Study of structure and mechanism of antibiotic action of antimicrobial peptide hedistin.The continued evolution of resistance to conventional antibiotics has led to wide range of consultation at national and international levels as to how to address this issue. There is growing consensus that a cornerstone requirement is the development of new antibiotics to help redress the balance of resistance versus available antibiotics. Antimicrobial peptides are considered as prospective antibiotic agents as they exhibit a broad spectrum of antimicrobial activities against Gram-positive, Gram-negative bacteria, fungi and so on.Hedistin is an antimicrobial peptide isolated from the coelomocytes of Nereis diversicolor, possessing activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus (Tasiemski,2007). However, the mechanism of antibiotic action of antimicrobial peptide hedistin is unclear. In our study, the three-dimensional structure of hedistin in both aqueous solution and deuterated dodecylphosphocholine (DPC) micelles was examined using nuclear magnetic resonance (NMR) techniques. And, the antibacterial process of hedistin was simulated using palmitoyl-oleoyl-phophatidylcholine (POPC) lipid bilayer and molecular dynamics (MD) simulation method. NMR results revealed that hedistin lacks secondary structure in aqueous solution, however, in DPC micelles, it features with a helix-turn-helix moiety and exhibits obvious amphipathic nature. The turn region (residues Val9-Thrl2) in the moiety is a four-residue hinge, lying in between the first N-terminal a-helix (residues Leu5-Lys8) and the second a-helix (residues Val13-Alal7) regions and causing an～120°angle between the axes of the two helices. MD results revealed that the segmental and nonlinear nature of hedistin structure is referred to as the heterogeneity of its helix-turn-helix motif which was found to be corresponding to a kind of discrete dynamics behavior, its dynamical heterogeneity, at the early stage of the MD simulations. That is, the first helix segment, prior to or following the second helix, binds to the lipid head-group region and subsequently permeates into the hydrophobic lipid tail region, and the hinge is the last portion entering the lipid environment. And, as the concentration of the peptide hedistin increases, the peptide molecules aggregate with each other and subsequently disrupt membrane cooperatively. When the peptide/lipid ratio is no less than 5/128, the strong disruption of peptide to membrane leads to the formation of a water pore in POPC membrane, which can explain the antibiotic process of antimicrobial peptide hedistin.2. Study of structure and mechanism of function of dihydrofolate reductase mutant DHFR-W21H.DHFR is an essential enzyme in cells, where it catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH as a coenzyme. An important residue in this reaction is Trp21 which is close to both the substrate and the coenzyme. Substitution of the tryptophan with a histidine residue results in a number of important observations. Firstly, NADPH binds to DHFR-W21H more than three orders of magnitude less than wild-type L.casei DHFR but has no obvious effect on the binding of the substrate FH2. Also the magnitude of the negative cooperative effect between NADPH and FH4 has a profile that reflects the cooperative ionization of two groups, one of which may belong to the new His21 residue. In view of these results we have embarked upon a series of NMR experiments that explore the structural and dynamics nature of this mutant. More specifically, the structures of the DHFR-W21H-MTX complex were examined at pH values 5.8 and 7.5 (above and below the His21 pKa). NMR results revealed that (1) the topology of DHFR-W21H at pH 5.8 and pH 7.5 are very similar to that of the wild-type DHFR, including eightβ-strands, four a-helix, and two long loops (residues 9-23 (A-B loop) and residues 119-135 (F-G loop)). (2) Slow transition of two conformations different in segment Pro20-His21-His22-Leu23 in DHFR-W21H-MTX complex at pH 5.8 and pH 7.5 was observed. (3) The 9-23 loop and F-G loop in DHFR-W21 H-MTX complex at pH 5.8 and pH 7.5 are more flexible than that in the wild-type complex. (4) Residue His21 in W21H is in involved in the catalysis process. These observations strongly indicate that the flexibility of residues 9-23 (A-B loop) and residues 119-135 (F-G loop) is related directly to the reactivity of the enzyme DHFR.