中文摘要
隨著細菌的抗藥性嚴重增加，抗菌胜肽的開發是一件迫切需要被研究的課題
這此篇論文中我們以S1 (KKWRKWLAKK)這條延伸自擁有良好殺菌能力的W5K/A9W (KKWRKWLKWLAKK) 為基礎，在S1上做些許修飾，這些修飾包含了把S1中全部的色胺酸換成側鏈較長且帶高疏水性的人工合成胺基酸Nal (D-β-naphthylalanine)；另一種修飾是在S1的末端依序加入一至三個Nal 形成一段帶有疏水性的尾端. 從實驗結果得知當S1中的色胺酸被換成Nal，有效增加胜肽的殺菌能力及降低溶血性。而在S1的末端添加Nal的數目越多，抗菌胜肽對鹽度的耐受性表現穩定進而有良好的殺菌能力表現。在高鹽環境中抗菌胜肽仍然保有殺菌能力，這個現象是之前的W5K/A9W及S1所沒有的特性，而這個Nal疏水性末端給予胜肽良好對鹽度的耐受度，進而讓抗菌胜肽在高鹽濃度的環境中能施展殺菌的能力。這個結果可廣泛應用在當抗菌胜肽有對鹽敏感的問題時在末端加上具疏水性的胺基酸可增加對鹽的忍受程度。本篇實驗結果提供一個改善抗菌胜肽在高鹽溶液下功能削減的缺點，使抗菌胜肽可在人體生理環境及任何高鹽環境中有更佳的開發應用潛能。

Abstract

With the increasing of antibiotic resistant pathogens, antimicrobial peptide is the optimal candidate for treating these pathogens. However, most of antimicrobial peptides reduced its ability under high salt concentration. Herein, we reported a new approach to diminish the influence of salinity for AMPs, through end tagging with the bulky aromatic amino acid, Nal (β-naphthylalanine) at the terminus of peptide. S1 (KKWRKWLAKK), a 10-mer peptide derived from W5K/A9W (KKWRKWLKWLAKK) originated from PEM2. Unfortunately, S1 lost its bactericidal performance, but hemolytic ability got a significant decline. Therefore, we hypothesized that the length of antimicrobial peptide affects the bactericidal function. In this study, adding several Nal at the terminus of S1 to form the hydrophobic end tagging. Determine the length of peptide or the end tagging, which is the pivotal one in determining the antimicrobial functions. The results indicated that end tagging length preserves antimicrobial ability of the peptide at high salinity. This result can be applied in any salt-sensitive antimicrobial peptide, conjugated a hydrophobic end tagging at the terminus can solve this problem. This easy strategy promote the antimicrobial peptide can exert its best effect on high salt condition, mammalian physiology or any salt-related situation.

REFERENCE
Avrahami D, Oren Z, Shai Y (2001) Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides. Biochemistry 40: 12591-12603

Baltzer SA, Brown MH (2011) Antimicrobial peptides: promising alternatives to conventional antibiotics. Journal of molecular microbiology and biotechnology 20: 228-235

Brender JR, McHenry AJ, Ramamoorthy A (2012) Does cholesterol play a role in the bacterial selectivity of antimicrobial peptides? Frontiers in immunology 3: 195

Brown KL, Poon GF, Birkenhead D, Pena OM, Falsafi R, Dahlgren C, Karlsson A, Bylund J, Hancock RE, Johnson P (2011) Host defense peptide LL-37 selectively reduces proinflammatory macrophage responses. J Immunol 186: 5497-5505

Chavez MI, Andreu C, Vidal P, Aboitiz N, Freire F, Groves P, Asensio JL, Asensio G, Muraki M, Canada FJ, Jimenez-Barbero J (2005) On the importance of carbohydrate-aromatic interactions for the molecular recognition of oligosaccharides by proteins: NMR studies of the structure and binding affinity of AcAMP2-like peptides with non-natural naphthyl and fluoroaromatic residues. Chemistry 11: 7060-7074

Cheng JT, Hale JD, Kindrachuk J, Jenssen H, Elliott M, Hancock RE, Straus SK (2010) Importance of residue 13 and the C-terminus for the structure and activity of the antimicrobial peptide aurein 2.2. Biophysical journal 99: 2926-2935

Ferguson N, Sharpe TD, Johnson CM, Schartau PJ, Fersht AR (2007) Structural biology: analysis of 'downhill' protein folding. Nature 445: E14-15; discussion E17-18

Fierz B, Kiefhaber T (2007) End-to-end vs interior loop formation kinetics in unfolded polypeptide chains. Journal of the American Chemical Society 129: 672-679

Garlapati S, Garg R, Brownlie R, Latimer L, Simko E, Hancock RE, Babiuk LA, Gerdts V, Potter A, van Drunen Littel-van den Hurk S (2012) Enhanced immune responses and protection by vaccination with respiratory syncytial virus fusion protein formulated with CpG oligodeoxynucleotide and innate defense regulator peptide in polyphosphazene microparticles. Vaccine 30: 5206-5214

Glukhov E, Stark M, Burrows LL, Deber CM (2005) Basis for selectivity of cationic antimicrobial peptides for bacterial versus mammalian membranes. The Journal of biological chemistry 280: 33960-33967

Hancock RE (1997) Peptide antibiotics. Lancet 349: 418-422

Hancock RE, Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature biotechnology 24: 1551-1557

Haug BE, Skar ML, Svendsen JS (2001) Bulky aromatic amino acids increase the antibacterial activity of 15-residue bovine lactoferricin derivatives. Journal of peptide science : an official publication of the European Peptide Society 7: 425-432

Huang ML, Shin SB, Benson MA, Torres VJ, Kirshenbaum K (2012) A comparison of linear and cyclic peptoid oligomers as potent antimicrobial agents. ChemMedChem 7: 114-122

Jiang Z, Vasil AI, Hale JD, Hancock RE, Vasil ML, Hodges RS (2008) Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides. Biopolymers 90: 369-383

Kachel K, Asuncion-Punzalan E, London E (1995) Anchoring of tryptophan and tyrosine analogs at the hydrocarbon-polar boundary in model membrane vesicles: parallax analysis of fluorescence quenching induced by nitroxide-labeled phospholipids. Biochemistry 34: 15475-15479

Lequin O, Ladram A, Chabbert L, Bruston F, Convert O, Vanhoye D, Chassaing G, Nicolas P, Amiche M (2006) Dermaseptin S9, an alpha-helical antimicrobial peptide with a hydrophobic core and cationic termini. Biochemistry 45: 468-480

Nguyen LT, Haney EF, Vogel HJ (2011) The expanding scope of antimicrobial peptide structures and their modes of action. Trends in biotechnology 29: 464-472

Pasupuleti M, Chalupka A, Morgelin M, Schmidtchen A, Malmsten M (2009) Tryptophan end-tagging of antimicrobial peptides for increased potency against Pseudomonas aeruginosa. Biochimica et biophysica acta 1790: 800-808

Richards FM, Lamed R, Wynn R, Patel D, Olack G (2000) Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: overview and feasibility: properties of diazirine as a precursor. Protein science : a publication of the Protein Society 9: 2506-2517

Rivier JE, Jiang G, Koerber SC, Porter J, Simon L, Craig AG, Hoeger CA (1996) Betidamino acids: versatile and constrained scaffolds for drug discovery. Proceedings of the National Academy of Sciences of the United States of America 93: 2031-2036

Rothstein DM, Spacciapoli P, Tran LT, Xu T, Roberts FD, Dalla Serra M, Buxton DK, Oppenheim FG, Friden P (2001) Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5. Antimicrobial agents and chemotherapy 45: 1367-1373

Saeed ZA, Huang SC, Coy DH, Jiang NY, Heinz-Erian P, Mantey S, Gardner JD, Jensen RT (1989) Effect of substitutions in position 12 of bombesin on antagonist activity. Peptides 10: 597-603

Stokes AL, Miyake-Stoner SJ, Peeler JC, Nguyen DP, Hammer RP, Mehl RA (2009) Enhancing the utility of unnatural amino acid synthetases by manipulating broad substrate specificity. Molecular bioSystems 5: 1032-1038

Stromstedt AA, Pasupuleti M, Schmidtchen A, Malmsten M (2009) Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence. Biochimica et biophysica acta 1788: 1916-1923

Tachi T, Epand RF, Epand RM, Matsuzaki K (2002) Position-dependent hydrophobicity of the antimicrobial magainin peptide affects the mode of peptide-lipid interactions and selective toxicity. Biochemistry 41: 10723-10731

Wang CW, Yip BS, Cheng HT, Wang AH, Chen HL, Cheng JW, Lo HJ (2009) Increased potency of a novel D-beta-naphthylalanine-substituted antimicrobial peptide against fluconazole-resistant fungal pathogens. FEMS yeast research 9: 967-970

Wei SY, Wu JM, Kuo YY, Chen HL, Yip BS, Tzeng SR, Cheng JW (2006) Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity. Journal of bacteriology 188: 328-334

Wimley WC (2010) Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS chemical biology 5: 905-917

Wu JM, Wei SY, Chen HL, Weng KY, Cheng HT, Cheng JW (2007) Solution structure of a novel D-naphthylalanine substituted peptide with potential antibacterial and antifungal activities. Biopolymers 88: 738-745

Yazdanbakhsh K, Kang S, Tamasauskas D, Sung D, Scaradavou A (2003) Complement receptor 1 inhibitors for prevention of immune-mediated red cell destruction: potential use in transfusion therapy. Blood 101: 5046-5052

Yin LM, Edwards MA, Li J, Yip CM, Deber CM (2012) Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions. The Journal of biological chemistry 287: 7738-7745

Yount NY, Bayer AS, Xiong YQ, Yeaman MR (2006) Advances in antimicrobial peptide immunobiology. Biopolymers 84: 435-458

Yu HY, Tu CH, Yip BS, Chen HL, Cheng HT, Huang KC, Lo HJ, Cheng JW (2011) Easy strategy to increase salt resistance of antimicrobial peptides. Antimicrobial agents and chemotherapy 55: 4918-4921

Zelezetsky I, Pag U, Sahl HG, Tossi A (2005) Tuning the biological properties of amphipathic alpha-helical antimicrobial peptides: rational use of minimal amino acid substitutions. Peptides 26: 2368-2376