Structure and Mechanism of Action of an Indolicidin Peptide Derivative with Improved Activity against Gram-positive Bacteria

Indolicidin, an antimicrobial peptide with a unique amino acid sequence (ILPWKWPWWPWRR-NH2) is found in bovine neutrophils. A derivative of indolicidin, CP10A, has alanine residues substituted for proline residues and has improved activity against Gram-positive organisms. Transmission electron microscopy of Staphylococcus aureus andStaphylococcus epidermidis treated with CP10A showed mesosome-like structures in the cytoplasm. The peptide at 2-fold the minimal inhibitory concentration did not show significant killing ofS. aureus ISP67 (a histidine, uridine, and thymidine auxotroph) but did show an early effect on histidine and uridine incorporation and, later, an effect on thymidine incorporation. Upon interaction with liposomes, detergents, and lipoteichoic acid, CP10A was shown by circular dichroism spectroscopy to undergo a change in secondary structure. Fluorescence spectroscopy indicated that the tryptophan residues were located at the hydrophobic/hydrophilic interface of liposomes and detergent micelles and were inaccessible to the aqueous quencher KI. The three-dimensional structure of CP10A in the lipid mimetic dodecylphosphocholine was determined using two-dimensional NMR methods and was characterized as a short, amphipathic helical structure, whereas indolicidin was previously shown to have an extended structure. These studies have introduced a cationic peptide with a unique structure and an ability to interact with membranes and to affect intracellular synthesis of proteins, RNA, and DNA. Indolicidin, an antimicrobial peptide with a unique amino acid sequence (ILPWKWPWWPWRR-NH2) is found in bovine neutrophils. A derivative of indolicidin, CP10A, has alanine residues substituted for proline residues and has improved activity against Gram-positive organisms. Transmission electron microscopy of Staphylococcus aureus andStaphylococcus epidermidis treated with CP10A showed mesosome-like structures in the cytoplasm. The peptide at 2-fold the minimal inhibitory concentration did not show significant killing ofS. aureus ISP67 (a histidine, uridine, and thymidine auxotroph) but did show an early effect on histidine and uridine incorporation and, later, an effect on thymidine incorporation. Upon interaction with liposomes, detergents, and lipoteichoic acid, CP10A was shown by circular dichroism spectroscopy to undergo a change in secondary structure. Fluorescence spectroscopy indicated that the tryptophan residues were located at the hydrophobic/hydrophilic interface of liposomes and detergent micelles and were inaccessible to the aqueous quencher KI. The three-dimensional structure of CP10A in the lipid mimetic dodecylphosphocholine was determined using two-dimensional NMR methods and was characterized as a short, amphipathic helical structure, whereas indolicidin was previously shown to have an extended structure. These studies have introduced a cationic peptide with a unique structure and an ability to interact with membranes and to affect intracellular synthesis of proteins, RNA, and DNA. dodecylphosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1,2-dioleoyl-sn-glycero-3-phosphotempocholine 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine 1-palmitoyl-2-stearoyl-(12-doxyl)-sn-glycero-3-phosphocholine minimal inhibitory concentration circular dichroism total correlation spectroscopy nuclear Overhauser effect (/enhancement) spectroscopy double quantum-filtered correlation spectroscopy Antimicrobial cationic peptides are ubiquitous in nature and are thought to be an important component in innate host defenses against infectious agents (1Hancock R.E.W. Falla T.J. Brown M. Adv. Microb. Physiol. 1995; 37: 136-175Google Scholar). There are four structural classes of cationic antimicrobial peptides; the disulfide-bonded β-sheet peptides, including the defensins; the amphipathic α-helical peptides such as the cecropins and melittins; the extended peptides, which often have a single amino acid predominating (e.g. indolicidin); and the loop-structured peptides like bactenecin (1Hancock R.E.W. Falla T.J. Brown M. Adv. Microb. Physiol. 1995; 37: 136-175Google Scholar). The initial interactions of some cationic peptides with Gram-negative bacteria are thought to involve binding to surface lipopolysaccharide (2Piers K.L. Hancock R.E.W. Mol. Microbiol. 1994; 12: 951-958Crossref PubMed Scopus (78) Google Scholar, 3Sawyer J.G. Martin N.L. Hancock R.E. Infect. Immun. 1988; 56: 693-698Crossref PubMed Google Scholar). The peptides displace divalent cations that are essential for outer membrane integrity and consequently distort the outer membrane bilayer (4Peterson A.A. Fesik S.W. McGroarty E.J. Antimicrob. Agents Chemother. 1987; 31: 230-237Crossref PubMed Scopus (58) Google Scholar). This allows access to the cytoplasmic membrane where peptide channel formation has been proposed to occur (5Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (584) Google Scholar). It is increasingly disputed as to whether peptide channel formation leads to dissolution of the proton motive force and leakage of essential molecules (6Cociancich S. Ghazi A. Hoffman J.A. Hetrus C. Letellier C. J. Biol. Chem. 1993; 268: 19239-19245Abstract Full Text PDF PubMed Google Scholar, 7Juretic D. Chan H.C. Brown J.H. Morell J.L. Hendler R.W. Westerhoff H. FEBS Lett. 1989; 249: 219-223Crossref PubMed Scopus (76) Google Scholar) or whether it is an intermediate step in the uptake of peptide into the cytoplasm, where it inhibits an essential function by e.g.binding to polyanionic DNA (8Wu M. Maier E. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 7235-7242Crossref PubMed Scopus (631) Google Scholar, 9Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (694) Google Scholar, 10Zhang L. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 8102-8111Crossref PubMed Scopus (134) Google Scholar). Indolicidin is a 13-amino acid cationic peptide present in the cytoplasmic granules of bovine neutrophils (11Selsted M.E. Novotny M.J. Morris W.L. Tang Y.Q. Smith W. Cullor J.S. J. Biol. Chem. 1992; 267: 4292-4295Abstract Full Text PDF PubMed Google Scholar). Indolicidin has a unique amino acid composition (ILPWKWPWWPWRR-NH2) with 39% tryptophan, 23% proline, and an amidated carboxyl terminus. This, along with the fact that it has a broad spectrum of antimicrobial activity, has made it an interesting candidate for study. The structure of indolicidin was determined by two-dimensional NMR and shown to form an extended boat-shaped conformation when bound to dodecylphosphocholine (DPC)1(12Rozek A. Friedrich C.L. Hancock R.E.W. Biochemistry. 2000; 39: 15765-15774Crossref PubMed Scopus (276) Google Scholar). Previous structure-function studies by Subbalakshmi et al. (13Subbalakshmi C. Krishnakumari V. Nagaraj R. Sitaram N. FEBS Lett. 1996; 395: 48-52Crossref PubMed Scopus (120) Google Scholar) led to the design of a peptide ILA, called here CP10A. CP10A is a peptide with the three proline residues of indolicidin replaced with alanine, resulting in the amino acid sequence ILAWKWAWWAWRR-NH2. Subbalakshmi et al. (13Subbalakshmi C. Krishnakumari V. Nagaraj R. Sitaram N. FEBS Lett. 1996; 395: 48-52Crossref PubMed Scopus (120) Google Scholar) found that these amino acid substitutions had no effect on the activity against Escherichia coli and Staphylococcus aureus (13Subbalakshmi C. Krishnakumari V. Nagaraj R. Sitaram N. FEBS Lett. 1996; 395: 48-52Crossref PubMed Scopus (120) Google Scholar). However, our previous studies show that CP10A has 2–8-fold better activity against most Gram-positive bacteria (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). In the present work we examined the structure and the mode of action of CP10A against Gram-positive bacteria in greater detail using a variety of biophysical and biochemical methods. Various studies of the effects of cationic peptides on the membranes of Gram-positive bacteria have been conducted. Previous studies of peptide effects on membrane potential show that there are effects on the cytoplasmic membrane of S. aureus (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar, 15Yeaman M.R. Bayer A.S. Koo S.P. Foss W. Sullam P.M. J. Clin. Invest. 1998; 101: 178-187Crossref PubMed Scopus (165) Google Scholar). As well, ultrastructural studies of S. aureus treated with defensins (16Shimoda M. Ohki K. Shimamoto Y. Kohashi O. Infect. Immun. 1995; 63: 2886-2891Crossref PubMed Google Scholar) and platelet microbicidal proteins (15Yeaman M.R. Bayer A.S. Koo S.P. Foss W. Sullam P.M. J. Clin. Invest. 1998; 101: 178-187Crossref PubMed Scopus (165) Google Scholar) showed cell membrane damage followed by cell death. The defensins caused mesosome-like structures to appear before the bacteria lost their viability, but no remarkable effects on the cell wall were seen (16Shimoda M. Ohki K. Shimamoto Y. Kohashi O. Infect. Immun. 1995; 63: 2886-2891Crossref PubMed Google Scholar). These studies suggested that membrane perturbation is an important, but not necessarily lethal, event. Recent studies indicate that some peptides may indeed have an intracellular target. Xiong et al. (17Xiong Y.Q. Yeaman M.R. Bayer A.S. Antimicrob. Agents Chemother. 1999; 43: 1111-1117Crossref PubMed Google Scholar) find that S. aureus, pretreated with inhibitors of DNA gyrase or protein synthesis, demonstrated decreased or blocked killing by human neutrophil peptide-1 and platelet microbicidal protein-1, whereas pretreatment with bacterial cell wall synthesis inhibitors enhanced bacterial killing. The authors concluded that these cytoplasmic membrane effects occurred before effects on protein and DNA synthesis. As well, in our lab we demonstrated a lack of correlation between bacterial killing and cytoplasmic membrane depolarization and, in Staphylococcus epidermidis, nuclear condensation, indicating effects on DNA (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). There is thus growing evidence for an intracellular target for some antimicrobial cationic peptides. Here we describe a peptide with a unique structure that causes ultrastructural effects on S. aureus and S. epidermidis similar to those seen with other cationic peptides as well as intracellular effects. CP10A (ILAWKWAWWAWRR-NH2) was synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry at the Nucleic Acid Protein Service (NAPS) unit at the University of British Columbia. The peptide was pure, as confirmed by high performance liquid chromatography and mass was S. aureus and S. epidermidis University of British M.R. Hancock R.E.W. Infect. Immun. 1999; PubMed Google Scholar) were for electron S. aureus ISP67 a for uridine, and histidine, was in the synthesis In most was as a with the ofS. aureus which was in amino and with uridine, and histidine (5Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (584) Google Scholar) was and were and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-stearoyl-(12-doxyl)-sn-glycero-3-phosphocholine were bacteria were treated with the peptide at the minimal inhibitory concentration for at This concentration was to an effect on a greater of the bacterial were with for The were in for with in a of and in The was were on and with and The and were was with a The of CP10A was determined using a the of D. V. in Scholar). of the peptide were made in bovine acid in well was with of the in or in the to a concentration of The was as the peptide concentration at which was of at The depolarization of the cytoplasmic membrane of S. aureus by the peptides was determined using the membrane (5Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (584) Google Scholar) A.S. Hoffman Biochemistry. PubMed Scopus Google Scholar) by a of the of et al. (8Wu M. Maier E. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 7235-7242Crossref PubMed Scopus (631) Google as in Friedrich et al. (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). of S. aureus ISP67 were in and to to at of The were and in with or of at CP10A was at and were at and and and to acid with to the on and at the were a on and with The were and into with liquid and on a to incorporation into the were in of and on to with to a A of lipid was with the peptide in This was a of in to the The resulting was in The was of to liposomes, followed by a using an Fluorescence were on an were between and at using a cell at The was to with the and to were by of the lipid or detergent The and lipid or detergent in The aqueous quencher was in to tryptophan to the aqueous were to the tryptophan in the were using a spectrum was the of using a cell of at The was at a step of and were peptides in The of lipid or detergent were or were by a spectrum of a the were to in of The of CP10A was by a of in to the peptide to of CP10A and The was to for the NMR were at and on a at L. J. Res. Scopus Google J. J. Chem. Scopus Google and M. K. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar) were was using the M. V. V. J. 1992; PubMed Scopus Google Scholar, V. M. R. V. J. Res. A. 1993; Scopus Google Scholar) or by the were with in and were using the sequence A. J. Res. Scholar) at a of were at of and of of CP10A in micelles at were determined by the peptide in at a of peptide to detergent of The was determined to be for the NMR were the dissolution in a spectrum with a total of was followed by spectrum to The NMR were with S. J. A. J. 1995; PubMed Scopus Google Scholar). was by of the with The were to at the before were using a were to NMR were using J. 1994; PubMed Scopus Google Scholar). were in the at using a of The were to which were using the of proton as well as between tryptophan The thus to which is well the was to a by and using the suggested by et al. Protein 1992; PubMed Scopus Google Scholar). were by and to the bound for and were by to the were using the and J. M. J. 1992; PubMed Scopus Google Scholar, M. FEBS Lett. 1988; PubMed Scopus Google Scholar, A. of the of by J. Scholar). structures by structures that had of were using and the force The of the to resulting in structures that with of and The was of S. epidermidis and S. aureus were to ultrastructural The bacteria were treated with of CP10A for before and for S. epidermidis and S. aureus, These electron showed the formation of intracellular membranes in these occurred the In to the peptide S. epidermidis (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google no nuclear was and there to be minimal cell wall effects with or leakage of cytoplasmic was have previously shown (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar) that CP10A had the and or 2–8-fold aureus, Staphylococcus aureus, S. epidermidis, with indolicidin against Gram-positive synthesis we determined the of CP10A on the S. aureus and the in the of and This was to that there are no significant between the The of CP10A against the was in and in the The of CP10A against the was in and in These showed that there were no significant in activity against the and in the CP10A had intracellular we synthesis S. aureus ISP67 was in synthesis in the of and the of CP10A. in with these showed that at 2-fold there were or the whereas at there were in the there was no significant between the in synthesis effects which demonstrated These indicated that the effects on synthesis at peptide were not the of or synthesis of RNA, and proteins did not as was membrane resulting in leakage of essential and uridine incorporation and to be with the the peptide incorporation was not These that CP10A had intracellular effects on S. of CP10A at 2-fold the and the on thymidine uridine and histidine incorporation into S. RNA, and protein CP10A had the ability to the cytoplasmic membrane ofS. aureus, we the membrane (5Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (584) Google Scholar). CP10A the membrane at a concentration of not These were similar to those found with the α-helical peptides and which the membrane at (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). In indolicidin and derivative the cytoplasmic membrane of aureus (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar) or E. coli (8Wu M. Maier E. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 7235-7242Crossref PubMed Scopus (631) Google Scholar). The conformation of CP10A was determined using NMR spectroscopy in the of it was important to that the structure of peptide binding to detergent micelles was similar to the structure when bound to which the bacterial cytoplasmic Fluorescence and spectroscopy are methods for the interaction of peptides in membrane The of CP10A in aqueous and bound to lipid and are shown in The of the peptide in was lipid an of the as well as an in indicating a of the tryptophan an aqueous to a of Fluorescence Google Scholar). The in and were with of indicating that the by the peptide in and was the aqueous quencher was in not no change in was the peptide was in the of liposomes, indicating that the tryptophan residues had inaccessible to the aqueous with were to the of the tryptophan residues in the as a in tryptophan when in In the of there was an in In the of and there was and The of a significant between the effects caused by and may be by the of tryptophan residues in CP10A. of the tryptophan may have the whereas were the It is that most of the tryptophan residues were located in an intermediate between the and resulting in similar effects for were found with the peptide indolicidin, which showed a in with and a for and A. Friedrich C.L. Hancock R.E.W. Biochemistry. 2000; 39: 15765-15774Crossref PubMed Scopus (276) Google Scholar). indolicidin and CP10A in the bilayer which is the of tryptophan Biol. 1996; PubMed Scopus Google Scholar). The of and CP10A are shown in A. The when bound to and lipoteichoic acid not similar with at and as well as a There was a seen in the of that not be in the of liposomes to by D. Biochem. PubMed Scopus Google Scholar). The double at and along with the at were of α-helical structure J. Chem. PubMed Scopus Google Scholar). The at was to the of tryptophan residues R.W. Biophys. J. 1994; PubMed Scopus Google Scholar). The of that the structure of CP10A in the of is with that seen in the of and The spectrum of CP10A in was the in liposomes, and indicating that binding of the peptide to as by the of the led to a secondary structure. In aqueous and the at and were with indicating that a helical structure is present in the of However, the at was not a tryptophan These indicated that CP10A a structure in the of The of the peptide indolicidin are shown for in CP10A was α-helical in the spectrum of indolicidin in was of or A. Friedrich C.L. Hancock R.E.W. Biochemistry. 2000; 39: 15765-15774Crossref PubMed Scopus (276) Google Scholar). The structure of CP10A was determined using proton nuclear methods. and were at and The proton were using the K. NMR of and Nucleic Google Scholar). The are in a of the spectrum the well of between the and as well as interactions the tryptophan The of occurred for and However, at these were for the of the The between the four residues were the with to the were were to and were for residues as well as residues and This is for a helical structure K. NMR of and Nucleic Google Scholar). present were between residues and which were the terminus. These are for a K. NMR of and Nucleic Google of CP10A in of peptide to at and in a The structure of CP10A was using and using methods. were to a well structure. The of structures is shown in A. The structure was well between residues and with to the of for and for The for residues were of an α-helical structure, with the of residues and to a helical The of was by the of against no of was the for residues was for the indicating The proton of residues or and were in the spectrum the of were structure However, in the were residues and for and residues and for The structures show between residues and as well as and the helical of CP10A. The was with the and residues on of the a whereas the tryptophan and alanine residues were on the other of the the The seen in the electron are similar to those seen with other peptides including defensins (16Shimoda M. Ohki K. Shimamoto Y. Kohashi O. Infect. Immun. 1995; 63: 2886-2891Crossref PubMed Google and with S. aureus (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). have been proposed to be a that is of some change or damage to the cytoplasmic occur with the and the T. in A on the of and E. J. Scholar). However, other peptides to cell wall effects and as well as nuclear with S. epidermidis, which was not for CP10A. electron of S. with CP10A showed no significant in the outer surface of the bacteria with the not The effects of peptides led to that peptides may have a of action that may (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). CP10A to have effects on the of the bacterial synthesis studies that intracellular effects occurred at peptide of synthesis did not occur as be cytoplasmic occurred that in of or of and uridine incorporation to be before thymidine incorporation. The of peptides in the of and in the of histidine, uridine, and thymidine were not that the were not as inhibitors of the peptides. the peptides did not with uptake into the cell and indeed synthesis. It is thus that CP10A the cytoplasmic membrane of S. aureus to an intracellular target. The unique sequence and activity of CP10A made it an interesting candidate for structural The spectrum of CP10A in aqueous was not that of a in to other α-helical cationic peptides such as and C. N. H. Hancock R.E. Antimicrob. Chemother. 1999; 43: PubMed Google Scholar). This was for such a However, interaction with the at and were indicating that there was a of helical structure In and detergent a at The was to the of tryptophan the at for helical structure, was by the at The of the were similar in the of liposomes, and the and The was not to the of α-helical structure that was seen with In the at to the tryptophan was not present in the interface by is for tryptophan and The of the in the of indicated a of the tryptophan residues into a The of was with using membrane that the tryptophan residues on were located in the membrane The tryptophan of the peptide in lipid membranes was not by the aqueous quencher indicating that these residues inaccessible to the The similar of CP10A in lipid and suggested that the tryptophan residues in CP10A were in a similar the tryptophan at in the were indicating a similar of tryptophan with to the The structure of CP10A in micelles determined NMR was α-helical with of helical to the have been suggested to be in the between a and helical in peptides S. 1999; PubMed Scopus Google Scholar, Biochemistry. 1995; PubMed Scopus Google Scholar). the of CP10A in aqueous were not of an structure, the and peptide may between a and an α-helical for which are The structural between indolicidin and CP10A were by the of the three proline residues with has the of amino acid which has been in to A. T. Protein 1994; PubMed Scopus Google Scholar). The of in CP10A, three and four residues may have had an these on the of the for the tryptophan the formation of the interaction of tryptophan with the membrane interface has a on the structure of CP10A. was found to have the of amino for the between the aqueous and the membrane interface Biol. 1996; PubMed Scopus Google Scholar). The including the of and of CP10A was well This be in to formation with the amidated terminus. between residues and as well as between and the were in the structures but not be confirmed by The of was between the of and whereas the of was This is with the of proton for these residues to effects. an interaction with the tryptophan the interactions are a form of interactions and have been as 1996; PubMed Scopus Google Scholar, J. Mol. Biol. 1994; PubMed Scopus Google Scholar). The tryptophan may the at the of CP10A and of the into the The change of structure in CP10A with indolicidin is for improved activity against Gram-positive In CP10A to have that were of other α-helical peptides our showed that CP10A was a better membrane indolicidin (14Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (408) Google Scholar). indicated that CP10A lipid similar to other helical peptides, whereas was not for It is interesting to that membrane depolarization by CP10A was not to of the bacterial as confirmed by our electron have characterized a unique CP10A, using biophysical and biochemical methods. microscopy showed minimal effects of the peptide on the cell membrane depolarization by CP10A was no nuclear was seen in the electron synthesis indicated that acid and protein were by the determined the structure and membrane of CP10A using and CP10A a helical conformation in the of which was present in aqueous The of CP10A was in the membrane which is the of the tryptophan interactions between the and tryptophan to peptide and may membrane our describe a structure that to the peptide to the cell wall and cytoplasmic membrane of Gram-positive bacteria and intracellular a growing of evidence that antimicrobial peptides may not by membrane but may of including intracellular The structure of CP10A may thus as a for the design of

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