Understanding Biology Using Peptides
Sylvie E. Blondelle (Editor)
American Peptide Society, 2005
Sample Manuscript: Just Replace This Text With Yours
Lenore M. Margoliath1, Khaled A. Farid2, Third A. Author3 and
Firstname L. Lastname4
1Department
of Cell and Molecular Biology; 2Department of Biomedical Sciences; 3Graduate
School of Oceanography; 4Department of Fisheries, Animal and Veterinary Sciences,
University of Rhode Island, Kingston, RI 02881, USA
Introduction
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Now back to the sample manuscript… Vibrio anguillarum infections result in
huge economic losses in the aquaculture industry annually. Pleurocidin, an
antimicrobial peptide (AMP) isolated in 1997 from winter flounder, exhibited MICs
in the micromolar range against a wide variety of fish and human pathogens [1].
Studies have shown a correlation between antimicrobial activity and the charges,
hydrophobicities, and hydrophobic moments of amphipathic peptides that are
capable of adopting an -helical structure [2]. Although the C-terminal acid is the
naturally-occurring form, synthetic pleurocidin amide is more potent and has been
shown to protect coho salmon from vibriosis in vivo [3]. No additional increase in
potency was obtained by adding a lysine at the N-terminus of pleurocidin amide to
increase the positive charge on the peptide [3]. To characterize the structural
determinants required for antimicrobial activity and to discover minimal sequences
for genetic engineering, we synthesized pleurocidin amide and two new pleurocidin
analogs LM4-1 and LM4-2 (see Figure 1). The analogs were responsible for helix
formation in cecropin A [4]. AMPs were screened against E.coli, V. anguillarum,
and V.carchariae.
Cecropin A
LM4-1
Pleurocidin
LM4-2
H-WKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK-NH2
H-GWGSFKKAAHVGKHVGK-NH2
H-GWGSFFKKAAHVGKHVGKAALTHYL-OH
H-GWGSFFKKAAHVGKHVGKAAL-NH2
Fig. 1. Sequences of cecropin A, native pleurocidin and synthetic pleurocidin analogs
Results and Discussion
Synthetic peptides pleurocidin amide (25 residues), LM4-1 ([des-F5]Pleurocidin 1-18
amide) and LM4-2 (pleurocidin 1-21 amide) were synthesized using Boc chemistry
on MBHA (1.1 mmol/g, 1g) resins in a CS-Bio peptide synthesizer. The N-terminal
Boc-group was left on during removal of Dnp from His residues using thiophenol in
DMF. Following low-high HF deprotection and cleavage with thiocresol and cresol
Table 1. Antibacterial activity of peptides
Minimum Bactericidal Concentration (µM)a
CSMHB
CSMHB + 1.5% NaCl
Peptide
V. anguillarum
E.coli (ZK4)
V. anguillarum
V. carchariae
Pleurocidin amide
25
100
100
100
LM4-1
>100
>200
>200
>100
LM4-2
100
200
>200
>100
Tachyplesin (control)
25
50
50
25
aMBC
is the minimum peptide conc. where bacterial growth was not observed at 21h
scavengers, the crude peptides were extracted from the resin with 10% aqueous
HOAc; material excluded from a Sephadex® G-25 column was pooled and
lyophilized, then dissolved in water, and analyzed by C18 RP-HPLC (Vydac
218TP54) with a gradient of 5-85% CH3CN in aq. 0.1% TFA.
Antibacterial assays were performed by evaluating the ability of synthetic
peptides to inhibit bacterial growth in cell cultures (Table 1). Just to show how to
insert another type of Figure, stereo picture is shown in Figure 2. Synthetic
pleurocidin amide exhibited similar antibiotic activity to tachyplesin, with the
desired activity against marine pathogens V. anguillarum and V. carchariae.
Pleurocidin was not as potent against E. coli as it was against V. anguillarum. The
pleurocidin analogs were an order of magnitude less active than the parent peptide,
and this result indicated that the C-terminus of pleurocidin might play a role in
stabilizing the helix. LM4-2 was more active than LM4-1, possibly indicating that
Phe6 is required for optimal helix formation and activity.
Fig. 1. A representative structure of GIDY. The ribbon shows the peptide backbone.
Acknowledgments
The work was funded by an USDA-NRICGP grant to L. Martin.
References
1. Cole, A. M., Weis, P. and Diamond, G. J. Biol. Chem. 272, 12008-12013 (1997).
2. Tossi, A., Sandri, L. and Giangaspero, A. Biopolymers 55, 4-30 (2000).
3. Jia, X., et al. Appl. Environ. Microbiol. 66, 1928-1932 (2000).
4. Merrifield, R. B., Vizioli, L. D. and Boman, H. G. Biochem. 21, 5020-5031 (1982).
Instructions:
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