CORD-19-Sentences  

CORD-19:111c3fadae9ebcddb5cef8c388db2a8f3fb55532 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-93 Sentence denotes pH-Dependent Lytic Peptides Discovered by Phage-Display † NIH Public Access Author Manuscript
TextSentencer_T2 95-103 Sentence denotes Abstract
TextSentencer_T3 104-215 Sentence denotes Lipid membranes compartmentalize eukaryotic cells and separate the cell interior from the extracellular milieu.
TextSentencer_T4 216-432 Sentence denotes So far, studies of peptide and protein interactions with membranes have largely been limited to naturally occurring peptides or to sequences designed on the basis of structural information and biophysical parameters.
TextSentencer_T5 433-630 Sentence denotes To expand on these studies, utilizing a system with minimal assumptions, we used phage-display technology to identify 12 amino-acid-long peptides that bind to liposomes at pH 5.0 but not at pH 7.5.
TextSentencer_T6 631-721 Sentence denotes Of the nineteen peptides discovered three were able to cause leakage of liposome contents.
TextSentencer_T7 722-847 Sentence denotes Multivalent presentation of these membrane-active peptides by conjugation onto poly(L-Lysine) enhanced their lytic potential.
TextSentencer_T8 848-1020 Sentence denotes Secondary structures were analyzed by circular dichroism in aqueous 2,2,2-trifluoroethanol and in buffered aqueous solutions, in both the presence and absence of liposomes.
TextSentencer_T9 1021-1155 Sentence denotes Two of the three lytic peptides show alpha helical profiles whereas none of the non-lytic peptides formed stable secondary structures.
TextSentencer_T10 1156-1443 Sentence denotes The diverse characteristics of the peptides identified in this study demonstrate that phage-displayed peptide library screens on lipid membranes result in the discovery of non-classical membrane-active peptides, whose study will provide novel insights into peptide-membrane interactions.
TextSentencer_T11 1444-1698 Sentence denotes Membranes play an integral role in biology by acting as compartmentalizing barriers and are involved in many biological processes such as signaling (1), membrane trafficking (2) including endo-and exocytosis (3), and viral cell entry and propagation (4).
TextSentencer_T12 1699-1848 Sentence denotes Much of our understanding of protein-membrane interactions has been gained from studying the interaction of viral fusion proteins with membranes (5).
TextSentencer_T13 1849-1972 Sentence denotes The study of the interaction of fusion peptides of enveloped viruses with membranes proved to be particularly fruitful (6).
TextSentencer_T14 1973-2206 Sentence denotes The knowledge gained by these studies has greatly increased our understanding of not only the role of these fusion peptides in membrane fusion but yielded important insights into the interaction of peptides with membranes in general.
TextSentencer_T15 2207-2371 Sentence denotes Furthermore, these studies allowed for the harnessing peptides for improved delivery of genetic material and membrane impermeable drugs into the cell cytoplasm (7).
TextSentencer_T16 2372-2514 Sentence denotes Despite the large number of membrane-active peptides known, no apparent consensus sequence or motif exists that governs membrane activity (8).
TextSentencer_T17 2515-2711 Sentence denotes Mechanistically, membrane disturbance can range from surface or interfacial effects such as the "carpet" model (9,10) membrane insertion (11), membrane fusion, pore formation, or membrane rupture.
TextSentencer_T18 2712-2720 Sentence denotes For each
TextSentencer_T19 2722-2797 Sentence denotes of these mechanisms the structure and length requirements may be different.
TextSentencer_T20 2798-3074 Sentence denotes Until now, regions of both cellular and viral proteins as well as peptides that interact with membranes have been discovered predominantly by deletion and substitution studies (12) , by homology (13) , by photolabeling studies (14) and by biophysical analyses (15) (16) (17) .
TextSentencer_T21 3075-3204 Sentence denotes A more encompassing approach is an experimental one aimed at identifying peptides without being constrained by current paradigms.
TextSentencer_T22 3205-3302 Sentence denotes In this study, we achieve the identification of lytic peptides with minimal a priori assumptions.
TextSentencer_T23 3303-3551 Sentence denotes By using a phage-display peptide library screen (18) for peptides with membrane affinity, we circumvent structural or sequence restrictions used in rational peptide design (19, 20) and avoid the limitations imposed by combinatorial chemistry (21) .
TextSentencer_T24 3552-3649 Sentence denotes To test the concept, we have chosen to identify peptides that are lytic in a pHdependent fashion.
TextSentencer_T25 3650-3824 Sentence denotes An understanding of pH-dependent lytic behavior will help us to understand better processes such as endosomal escape of viruses and gene delivery vehicles into the cytoplasm.
TextSentencer_T26 3825-3984 Sentence denotes The knowledge gained by these studies opens the possibility to develop methods in interfering with this step in the infectious pathway of pathological viruses.
TextSentencer_T27 3985-4101 Sentence denotes Furthermore, the insights gained will pave the way to increase endosomal escape of non-viral gene delivery vehicles.
TextSentencer_T28 4102-4202 Sentence denotes The Ph.D.-12 Phage-display Peptide Library Kit was purchased from New England Biolabs (Beverly, MA).
TextSentencer_T29 4203-4270 Sentence denotes All lipids were purchased from Avanti Polar Lipids (Alabaster, AL).
TextSentencer_T30 4271-4679 Sentence denotes Cterminally amidated peptides were either purchased as crude powder from Research Genetics (Invitrogen, Carlsbad, CA) with the exception of INF7*, which was purchased from Anaspec (San Jose, CA), or synthesized using the FastMocTM chemistry with Fmoc-Rink amide MBHA resin 1 (Anaspec, San Jose, CA) on an Applied Biosystems (Foster City, CA) 431A peptide synthesizer using the protocol of Fisher et al (22) .
TextSentencer_T31 4680-4811 Sentence denotes Cleavage from the resin and deprotection was accomplished with a mixture of trifluoroacetic acid (TFA) : phenol: water: thioanisol:
TextSentencer_T32 4812-4869 Sentence denotes 1,2ethanedithiol (EDT): triisopropylsilane (TIPS) = 77.5:
TextSentencer_T33 4870-4872 Sentence denotes 5:
TextSentencer_T34 4873-4875 Sentence denotes 5:
TextSentencer_T35 4876-4878 Sentence denotes 5:
TextSentencer_T36 4879-4883 Sentence denotes 2.5:
TextSentencer_T37 4884-4886 Sentence denotes 5.
TextSentencer_T38 4887-5039 Sentence denotes Peptide synthesis grade reagents were purchased from Applied Biosystems, and resin and protected amino acids were purchased from Anaspec (San Jose, CA).
TextSentencer_T39 5040-5155 Sentence denotes Poly(L-Lysine) hydrobromide (pLLys) was purchased from Sigma (St. Louis, MO), catalog number P2636, Mw = 30∼70 kDa.
TextSentencer_T40 5156-5235 Sentence denotes N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was purchased from Aldrich.
TextSentencer_T41 5236-5305 Sentence denotes Calcein (Sigma, St. Louis, MO) was used without further purification.
TextSentencer_T42 5306-5371 Sentence denotes All other chemicals were of reagent grade unless otherwise noted.
TextSentencer_T43 5372-5566 Sentence denotes Multilamellar vesicles were prepared from either 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) or from a mixture of lipids representing the endosomal membrane (endosomal lipid mix: ELM) (23) .
TextSentencer_T44 5567-5696 Sentence denotes The endosomal lipid mix consists of liver PC: liver PE: brain PS: brain SM: cholesterol: liver PI: porcine brain ganglioside = 5:
TextSentencer_T45 5697-5699 Sentence denotes 1:
TextSentencer_T46 5700-5702 Sentence denotes 1:
TextSentencer_T47 5703-5705 Sentence denotes 1:
TextSentencer_T48 5706-5708 Sentence denotes 3:
TextSentencer_T49 5709-5711 Sentence denotes 1:
TextSentencer_T50 5712-5714 Sentence denotes 1.
TextSentencer_T51 5715-5833 Sentence denotes 1.5 μmol of lipid was dried into a film under argon, and residual solvents were removed under high vacuum for an hour.
TextSentencer_T52 5834-6039 Sentence denotes The lipid film was then dispersed in 500 μl pH 5 buffer (25 mM citric acid, 25 mM Na 2 HPO 4 , 150 mM sodium chloride, pH 5 with sodium hydroxide) with 10% fetal bovine serum (FBS, Mediatech, Herndon, VA).
TextSentencer_T53 6040-6107 Sentence denotes The serum was added in the buffer to mimic cell culture conditions.
TextSentencer_T54 6108-6216 Sentence denotes After vortexing for 4 minutes, the MLVs were collected by centrifugation for 4 minutes at 20,800 × g at 4°C.
TextSentencer_T55 6217-6351 Sentence denotes The supernatant was removed and the MLVs were resuspended in 500 μl 10% FBS in pH 5 buffer for immediate use in screening experiments.
TextSentencer_T56 6352-6388 Sentence denotes The screen was conducted as follows:
TextSentencer_T57 6389-6516 Sentence denotes 2.4×10 11 plaque-forming units (PFU) were diluted with pH 5 buffer with 10% FBS, which was added to prevent unspecific binding.
TextSentencer_T58 6517-6639 Sentence denotes To remove any traces of potential phage aggregates, the phage solution was centrifuged for 4 minutes at 20,800 × g at 4°C.
TextSentencer_T59 6640-6704 Sentence denotes The supernatant was added to the MLVs to a final volume of 1 ml.
TextSentencer_T60 6705-6890 Sentence denotes After incubation for 15 min at 37°C, with occasional shaking, those phages that bound to MLVs were harvested by collecting the MLVs by centrifugation for 4 minutes at 20,800 × g at 4°C.
TextSentencer_T61 6891-6999 Sentence denotes The supernatant was removed by aspiration, and the pellet was resuspended in 1 ml of 10% FBS in pH 5 buffer.
TextSentencer_T62 7000-7054 Sentence denotes This washing procedure at pH 5 was repeated ten times.
TextSentencer_T63 7055-7172 Sentence denotes After the final centrifugation, the MLV/phage pellet was resuspended in 1 ml 10% FBS in pH 7.5 buffer (pH 7.5 buffer:
TextSentencer_T64 7173-7265 Sentence denotes 25 mM citric acid, 25 mM Na 2 HPO 4 , 150 mM sodium chloride, pH 7.5 with sodium hydroxide).
TextSentencer_T65 7266-7460 Sentence denotes Following a 15 min incubation at 37°C, the phages that bound MLVs at pH 5, but were released from MLVs at pH 7.5, were harvested by removing the MLVs by centrifugation (10 min, 20,800 × g, 4°C).
TextSentencer_T66 7461-7530 Sentence denotes This supernatant was transferred into a fresh tube for amplification.
TextSentencer_T67 7531-7616 Sentence denotes The amplification and titering steps were performed as described by the manufacturer.
TextSentencer_T68 7617-7815 Sentence denotes After four rounds of screening, the phages were harvested and plaques were picked for sequencing (Department of Human Genetics, Mount Sinai School of Medicine) using the primers provided in the kit.
TextSentencer_T69 7816-8004 Sentence denotes The crude peptides were analyzed and purified on a Gilson HPLC system using reverse phase Vydac Protein and Peptide C18 analytical and semi-preparative columns (Grace Vydac, Hesperia, CA).
TextSentencer_T70 8005-8169 Sentence denotes The aqueous mobile phase consisted of 0.1% TFA or ammonium acetate in MilliQ water and the organic phase of 0.1% TFA or ammonium acetate in HPLC grade acetonitrile.
TextSentencer_T71 8170-8295 Sentence denotes The identity of the peptide was confirmed by MALDI-TOF (Rockefeller University, Protein DNA Technology Center, New York, NY).
TextSentencer_T72 8296-8414 Sentence denotes Peptides were conjugated onto SPDP activated poly(L-Lysine) (pLLys) using a method described by Erbacher et al. (24) .
TextSentencer_T73 8415-8500 Sentence denotes The fractions containing activated pLLys were then pooled and dialyzed against water.
TextSentencer_T74 8501-8643 Sentence denotes Activated pLLys was purified by gel permeation chromatography on a 1.25 cm × 45 cm Sephadex G25 column equilibrated in 0.15 M sodium chloride.
TextSentencer_T75 8644-8822 Sentence denotes The concentration of pLLys was determined by ninhydrin assay (45) , and the grade of modification was measured by thiopyridone release at 343 nm after incubation with excess DTT.
TextSentencer_T76 8823-9082 Sentence denotes Activated pLLys in one tenth the total reaction volume (2.2 ml) was added dropwise to peptides dissolved in 5 × final reaction buffer (2 M guanidine hydrochloric acid, 20 mM ammonium bicarbonate), layered with argon, and rotated at room temperature overnight.
TextSentencer_T77 9083-9146 Sentence denotes The extent of reaction was analyzed by thiopyridone absorbance.
TextSentencer_T78 9147-9453 Sentence denotes The peptide-pLLys conjugate was diluted 4-fold with 25 mM HEPES sodium hydroxide pH 7.4 and purified by ion-exchange chromatography on a 1 ml HiPrep S column (equilibrated in 25 mM HEPES sodium hydroxide pH 7.4, 0.5 M guanidine hydrochloric acid) using a linear gradient to 3 M guanidine hydrochloric acid.
TextSentencer_T79 9454-9511 Sentence denotes Elution profiles were monitored by ninhydrin assay (45) .
TextSentencer_T80 9512-9687 Sentence denotes After dialysis against 25 mM HEPES-sodium hydroxide pH 7.4, 150 mM sodium chloride, the final concentration of pLLys-peptide conjugate was determined by ninhydrin assay (45) .
TextSentencer_T81 9688-9805 Sentence denotes The final peptide concentration was determined by analytical HPLC after treatment with DTT and ninhydrin assay (45) .
TextSentencer_T82 9806-9970 Sentence denotes 250 μM of synthetic peptide was incubated with 2.5 mM MLVs of one of two lipid compositions, in a total volume of 100 μl in either pH 5 or 7.5 buffer with 1 mM DTT.
TextSentencer_T83 9971-10175 Sentence denotes After incubation for 30 minutes at 37°C, peptide bound to liposomes was pelleted by centrifugation for 4°C for 10 minutes at 20,800 × g followed by two washes with 200 μl of the respective chilled buffer.
TextSentencer_T84 10176-10303 Sentence denotes The pellets were dissolved in TFE and applied onto a thin layer chromatography plate (Silica Gel 60 F 254 , EMD Chemicals, Inc.
TextSentencer_T85 10304-10324 Sentence denotes Darmstadt, Germany).
TextSentencer_T86 10325-10451 Sentence denotes Peptide and lipids were resolved by thin layer chromatography with the mobile phase (1-butanol : acetic acid : water = 5:2:3).
TextSentencer_T87 10452-10582 Sentence denotes Peptides were visualized by ninhydrin spraying (1 mg/ml ninhydrin in water-saturated n-butanol) and heating for color development.
TextSentencer_T88 10583-10769 Sentence denotes Large unilamellar vesicles (LUVs) were prepared by extrusion using a Liposofast hand-held extruder ((25), Avestin, Ottawa, Canada) with 100 nm pore-sized polycarbonate filters (Avestin).
TextSentencer_T89 10770-10970 Sentence denotes Briefly, 25 μmol of lipid and 1 μCi of L-1,2-dipalmitoyl, 3-phosphatidyl [Nmethyl-3 H] choline ( 3 H-DPPC, Amersham Biosciences, Piscataway, NJ) in chloroform was dried into a film as described above.
TextSentencer_T90 10971-11127 Sentence denotes Liposomes for contents-leakage assays were prepared by hydrating the above film in 500 μl of calcein solution (40 mM calcein in pH 7.5 buffer) by vortexing.
TextSentencer_T91 11128-11336 Sentence denotes The lipid dispersion was then either sonicated to make SUVs (two-times-5 minutes continuous sonication at setting 10 with a Sonicator XL, Misonix, Farmingdale, NY) or extruded to make LUVs as described above.
TextSentencer_T92 11337-11482 Sentence denotes Free calcein was removed from liposomes by floatation on an Accudenz (Accurate Chemical & Scientific Corporation, Westbury, NY) density gradient.
TextSentencer_T93 11483-11531 Sentence denotes All Accudenz solutions are w/v in pH 7.5 buffer.
TextSentencer_T94 11532-11693 Sentence denotes 100 μl liposome solution was mixed with 100 μl 40% Accudenz and introduced into 5 × 41 mm ultra-Clear ultracentrifuge tubes (Beckman Instruments, Palo Alto, CA).
TextSentencer_T95 11694-11894 Sentence denotes This solution was then overlaid in succession with 150 μl 10%, 150 μl 5%, and 100 μl 0% Accudenz for POPC liposomes and 150 μl 15%, 150 μl 12.5%, 37.5 μl 10%, and 100 μl 5% Accudenz for ELM liposomes.
TextSentencer_T96 11895-12053 Sentence denotes Liposomes were centrifuged for 4 hours at 4°C at 48,000 RPM with deceleration set at 5 on the Beckman L8-M ultracentrifuge in an SW55Ti swinging bucket rotor.
TextSentencer_T97 12054-12122 Sentence denotes Calcein-loaded liposomes were isolated from the 5% and 0% interface.
TextSentencer_T98 12123-12261 Sentence denotes Lipid concentration was monitored by the amount of tritiated DPPC (Wallac 1409, Perkin-Elmer, Boston, MA; CytoScint, ICN, Costa Mesa, CA).
TextSentencer_T99 12262-12361 Sentence denotes Alternatively, liposomes can be purified by repeated spin-column chromatography using Sephadex G25.
TextSentencer_T100 12362-12480 Sentence denotes As originally described by Duzgunes et al. (26) , contents leakage was measured by calcein release from the liposomes.
TextSentencer_T101 12481-12597 Sentence denotes Peptide stocks were made in DMF and borate buffer pH 8.4 mixtures to allow for complete dissolution of all peptides.
TextSentencer_T102 12598-12664 Sentence denotes Peptide concentrations were determined by a ninhydrin assay (27) .
TextSentencer_T103 12665-12720 Sentence denotes Lipid concentrations were 25 μM unless otherwise noted.
TextSentencer_T104 12721-12829 Sentence denotes Final concentrations of stock components for leakage experiments were 1% DMF and 1.2 mM borate respectively.
TextSentencer_T105 12830-13053 Sentence denotes Assays were conducted in 96 well plates (Nunc white polysorb plates, Nalgene Nunc International, Rochester, NY), and calcein fluorescence was followed in a fluorescence plate reader (fmax, Molecular Devices, Sunnyvale, CA).
TextSentencer_T106 13054-13133 Sentence denotes Excitation and emission wavelengths were set to 485 nm and 538 nm respectively.
TextSentencer_T107 13134-13252 Sentence denotes Fluorescence was measured in 1 minute intervals with an integration time of 100 msec using SoftMax Pro 1.3.2 software.
TextSentencer_T108 13253-13399 Sentence denotes The reaction time was 30 min at 37°C, after which complete leakage was determined by the addition of Triton X100 to a final concentration of 0.2%.
TextSentencer_T109 13400-13483 Sentence denotes Percent leakage and leakage units were calculated as defined by Plank et al. (28) .
TextSentencer_T110 13484-13637 Sentence denotes Calcein leakage studies conducted with peptide-pLLys conjugates were normalized for peptide concentration as determined above, with and without 1 mM DTT.
TextSentencer_T111 13638-13832 Sentence denotes Peptide conformation was studied by circular dichroism (CD) spectroscopy using a JASCO J-810 CD spectrometer with a 1 mm path length quartz cuvette (Hellma QS 284, Hellma U.S.A., Plainview, NY).
TextSentencer_T112 13833-13941 Sentence denotes All spectra are averages of four measurements taken between 250 to 190 nm at 25±2°C, unless noted otherwise.
TextSentencer_T113 13942-14005 Sentence denotes Peptide stock solutions were prepared in trifluroethanol (TFE).
TextSentencer_T114 14006-14070 Sentence denotes Measurements were performed in TFE : water = 1:1 with 1 mM TCEP.
TextSentencer_T115 14071-14105 Sentence denotes The pH of these solutions was 3.0.
TextSentencer_T116 14106-14228 Sentence denotes For measurements of peptides in aqueous solution, peptides were dissolved in 0.1% ammonia, from which dilutions were made.
TextSentencer_T117 14229-14368 Sentence denotes Final CD buffer was 0.01% ammonia, 2 mM citrate, 2 mM borate, 2 mM potassium phosphate buffered, with 10 mM sodium chloride and 10 mM TCEP.
TextSentencer_T118 14369-14450 Sentence denotes The buffering components were used to adjust the solutions to either pH 5 or 7.5.
TextSentencer_T119 14451-14578 Sentence denotes If present, liposomes (SUVs), made as described above, were added to the peptide solution in less than 10% of the total volume.
TextSentencer_T120 14579-14709 Sentence denotes Peptide concentrations were derived from amino acid analysis (Rockefeller University Protein DNA Technology Center, New York, NY).
TextSentencer_T121 14710-14840 Sentence denotes Percent alpha helix was calculated taking the molar ellipticity at 222 nm with -32 × 10 3 deg·cm 2 ·dmol -1 defined as 100% (29) .
TextSentencer_T122 14841-15062 Sentence denotes The probability that a peptide sequence occurred by chance, and positional amino acid frequencies were calculated using algorithms in RELIC: INFO and AAFREQ (which was modified so peptide redundancies were allowed) (30) .
TextSentencer_T123 15063-15188 Sentence denotes The unamplified Ph.D.-12 library sequences were obtained from http://relic.bio.anl.gov/relicPeptides.aspx (file ran-12s.txt).
TextSentencer_T124 15189-15365 Sentence denotes Normalized amino acid occurrences for Fig. 4 were calculated with this data; namely, the frequency of a residue found in our phage screen divided by that of the parent library.
TextSentencer_T125 15366-15491 Sentence denotes The probability that amino acid X occurs at position y by chance in n out of N picked phage clones was calculated as follows:
TextSentencer_T126 15492-15596 Sentence denotes where F Ph.D.-12 (X at y) is the frequency of X at position y found in the unamplified Ph.D.-12 library.
TextSentencer_T127 15597-15704 Sentence denotes BLAST searches were done on NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) for short nearly exact matches.
TextSentencer_T128 15705-15845 Sentence denotes Significant motifs were not found within the discovered peptides according to MEME·MAST (http://meme.sdsc.edu/meme/website/intro.html) (31).
TextSentencer_T129 15846-15920 Sentence denotes All viruses have to breach a cellular membrane to access to the cytoplasm.
TextSentencer_T130 15921-16005 Sentence denotes Inevitably, this requires the interaction of viral proteins with cellular membranes.
TextSentencer_T131 16006-16152 Sentence denotes These interactions can either occur at the plasma membrane (e.g., HIV (32)) or in the endosomal system (e.g., influenza (33) and adenovirus (34)).
TextSentencer_T132 16153-16316 Sentence denotes Conformational changes of both class I and class II fusion proteins of enveloped viruses (35) play an essential role in fusion of the viral and cellular membranes.
TextSentencer_T133 16317-16478 Sentence denotes It has been recognized early on that pH-dependent interaction of fusion peptides with lipid bilayers are an important part of merging of membranes (12, 36, 37) .
TextSentencer_T134 16479-16697 Sentence denotes That pH-dependent membrane interactions are also vital in endosomal escape of non-enveloped viruses is demonstrated by the pH-dependent insertion of the capsid protein of rhinoviruses into the endosomal membrane (38) .
TextSentencer_T135 16698-16880 Sentence denotes Because of our interest in finding regions of other non-enveloped viruses involved in endosome escape, we set out to identify peptides that act on membranes in a pH-dependent manner.
TextSentencer_T136 16881-17117 Sentence denotes While different mechanisms can be envisaged that results in such pH-dependency, we reasoned that one possible basis for pH-dependent membrane activity is the ability of peptides to bind to membranes only at acidic but not at neutral pH.
TextSentencer_T137 17118-17218 Sentence denotes Thus, the screen was designed to identify peptides that bind to membranes at pH 5 but not at pH 7.5.
TextSentencer_T138 17219-17398 Sentence denotes Peptides with pH-dependent membrane binding properties were discovered by panning a phage-displayed peptide library against multilamellar vesicles (MLVs) at the two pH conditions.
TextSentencer_T139 17399-17539 Sentence denotes For our screening, the library Ph.D.-12 (NEB, Beverly, MA), which is composed of 2.7 × 10 9 random combinations of 12 amino acids, was used.
TextSentencer_T140 17540-17675 Sentence denotes The peptides in this library are linked by a GGGS spacer to the Nterminus of the minor coat protein pIII of the filamentous phage M-13.
TextSentencer_T141 17676-17755 Sentence denotes Specifically, the library of filamentous phage was incubated with MLVs at pH 5.
TextSentencer_T142 17756-17896 Sentence denotes Phages that bind stably to MLVs were then isolated by collecting the MLVs by centrifugation; non-binding phages remained in the supernatant.
TextSentencer_T143 17897-18012 Sentence denotes Those phages that did not bind firmly to MLVs at pH 7.5 were selected by eluting phages with pH 7.5 buffer at 37°C.
TextSentencer_T144 18013-18106 Sentence denotes This eluant was amplified to repeat the panning three more times (see Materials and Methods).
TextSentencer_T145 18107-18404 Sentence denotes Two different lipid compositions were used in this screen to select for peptides that either interact with the aliphatic bilayer core (using electroneutral PC liposomes) or the polar headgroups of the endosomal membrane (using an acidic endosomal lipid mix (ELM) described in the literature (23)).
TextSentencer_T146 18405-18497 Sentence denotes These experiments yielded nineteen distinct peptide sequences, which are listed in Table 1 .
TextSentencer_T147 18498-18565 Sentence denotes Some peptides occurred multiple times after four rounds of panning.
TextSentencer_T148 18566-18935 Sentence denotes This is not a result of the incomplete sequence representation or the positional amino acid biases dictated by M13 phage biology of the parent Ph.D.-12 library (39), nor individual phage growth biases, as the number of occurrences in the screen does not correlate with the probabilities of the sequences to randomly occur in the parent library (Supplementary Table 1 ).
TextSentencer_T149 18936-19043 Sentence denotes Overall, eleven short peptides were characterized for their ability to interact with and disturb membranes.
TextSentencer_T150 19044-19211 Sentence denotes Of the discovered peptides, all of the peptides recovered from the POPC MLV screen and the three peptides that occurred more than once in the ELM MLV screen were used.
TextSentencer_T151 19212-19353 Sentence denotes As positive controls for membrane activity two peptides were chosen that are known to be membrane-active in a pH-dependent manner (Table 1) .
TextSentencer_T152 19354-19596 Sentence denotes INF7 is the first 23 amino acids of the amino-terminal sequence of influenza virus X-31 (H3N2) hemagglutinin subunit HA-2 with two G to E substitutions (positions 4 and 7) that render the peptide more membrane-active in in vitro assays (28) .
TextSentencer_T153 19597-19672 Sentence denotes The other peptide is a 15-mer known as short fusion peptide 3 (SFP3) (40) .
TextSentencer_T154 19673-19846 Sentence denotes This amphipathic peptide is a shortened version of the synthetic pH-sensitive peptide GALA (41), which has been described in detail for its membrane activity in vitro (20) .
TextSentencer_T155 19847-20007 Sentence denotes In addition to the relevant amino acid sequences, a cysteine was added to the C-terminus of each peptide (except SFP3, which already has a C-terminal cysteine).
TextSentencer_T156 20008-20075 Sentence denotes Each peptide is named after the screen from which it was recovered.
TextSentencer_T157 20076-20130 Sentence denotes INF7 with the C-terminal Cysteine is now called INF7*.
TextSentencer_T158 20131-20258 Sentence denotes This additional cysteine allowed the conjugation of the peptides to poly-lysine for multimeric presentation as discussed later.
TextSentencer_T159 20259-20287 Sentence denotes The C-termini were amidated.
TextSentencer_T160 20288-20415 Sentence denotes The 13-mer peptides were first tested for whether they retained the ability to bind to liposomes on which the screen was based.
TextSentencer_T161 20416-20571 Sentence denotes To demonstrate peptide association with MLVs individual peptides were incubated with POPC or ELM MLVs under the same pH conditions as for the phage screen.
TextSentencer_T162 20572-20719 Sentence denotes After centrifugation, peptides and lipids in the pellet were separated by thin layer chromatography and the peptides visualized by ninhydrin spray.
TextSentencer_T163 20720-20796 Sentence denotes For optimal recovery and detection 250 μM peptide and 2.5 mM lipid was used.
TextSentencer_T164 20797-20956 Sentence denotes The peptides ELM1, ELM2, ELM3, PC3, PC5, and PC6 were, in contrast to PC1, PC2 and PC4, not detectable in the pellet of POPC MLVs (Table 2 and data not shown).
TextSentencer_T165 20957-21049 Sentence denotes For technical reasons it was not possible to measure the binding of PC2 and PC4 to ELM MLVs.
TextSentencer_T166 21050-21129 Sentence denotes Of the remaining peptides, however, only PC1 could bind to ELM MLVs (Table 2 ).
TextSentencer_T167 21130-21339 Sentence denotes PC4 pelleted also in the absence of MLVs in a pH-independent manner, although to a lesser degree than in their presence, suggesting that PC4 has a tendency to aggregate under these conditions (data not shown).
TextSentencer_T168 21340-21504 Sentence denotes To analyze the lytic potential of our peptides the ability of these peptides to induce leakage of vesicle contents of large unilamellar liposomes (LUVs) was tested.
TextSentencer_T169 21505-21589 Sentence denotes To measure liposome leakage a well-established contents leakage assay (26) was used.
TextSentencer_T170 21590-21794 Sentence denotes In this assay, the fluorophore calcein is entrapped in the liposomes at self-quenching concentrations; when released into the surrounding buffer, quenching is relieved resulting in increased fluorescence.
TextSentencer_T171 21795-21912 Sentence denotes A typical calcein release curve during incubation of 25 μM PC1 peptide with POPC LUVs at 37°C can be seen in Fig. 1 .
TextSentencer_T172 21913-22029 Sentence denotes As expected, due to substrate (i.e., non-lysed, calcein filled liposomes) depletion, the leakage curve was biphasic.
TextSentencer_T173 22030-22134 Sentence denotes At pH 5 the initial release rate was 4.2% leakage/min and the extent of leakage at 30 minutes was 34.5%.
TextSentencer_T174 22135-22188 Sentence denotes At pH 7.5, on the other hand, leakage was negligible.
TextSentencer_T175 22189-22440 Sentence denotes To determine the relative leakage activity of each peptide, contents leakage was determined at peptide concentrations from 0 to 75 μM (except for PC4, for which the highest concentration was 8.3 μM due to peptide aggregation at higher concentrations).
TextSentencer_T176 22441-22572 Sentence denotes Whereas the peptide concentrations were varied as a three-fold dilution series, the lipid concentration was kept constant at 25 μM.
TextSentencer_T177 22573-22660 Sentence denotes The titration curves for all the peptides analyzed by this method are shown in Fig. 2 .
TextSentencer_T178 22661-22839 Sentence denotes To summarize the leakage data from various concentrations of all peptides, their activity was represented in leakage units as defined previously by Plank et al. (Table 3 ) (28) .
TextSentencer_T179 22840-22983 Sentence denotes Leakage units are defined as the inverse of the amount of peptide (expressed in μg) necessary to result in ≥50% leakage after 30 minutes (28) .
TextSentencer_T180 22984-23076 Sentence denotes In effect, leakage units represent a molar leakage coefficient corrected for peptide length.
TextSentencer_T181 23077-23315 Sentence denotes As anticipated, some peptides PC1, PC2, and PC4 -namely those which bound to MLVsas well as the two positive control peptides SFP3 and INF7* demonstrate membrane activity with greater leakage at pH 5 than at pH 7.5 ( Fig. 2 and Table 3 ).
TextSentencer_T182 23316-23409 Sentence denotes The other peptides analyzed do not show any activity under these conditions (data not shown).
TextSentencer_T183 23410-23511 Sentence denotes The leakage triggered by INF7* is comparable to the results reported by Plank et al. (28) (Table 3 ).
TextSentencer_T184 23512-23591 Sentence denotes PC4 is almost as active as INF7* while PC2 is approximately 3-fold less active.
TextSentencer_T185 23592-23732 Sentence denotes The leakage activities of SFP3 and PC1 at pH 5 are more than one order of magnitude lower than the activity of INF7* (Table 3 and Fig. 2 ).
TextSentencer_T186 23733-23889 Sentence denotes For certain peptides and pH conditions it was not possible to calculate leakage units because 50% leakage was not reached in the concentration range tested.
TextSentencer_T187 23890-23973 Sentence denotes For two of these conditions, namely PC1 and SFP3 at pH 7.5, leakage was negligible.
TextSentencer_T188 23974-24187 Sentence denotes For the third condition, PC4 at pH 7.5, leakage measured at the highest peptide concentration without detectable peptide aggregation reached 27% (Fig. 2) , approximately 3-fold lower than leakage observed at pH 5.
TextSentencer_T189 24188-24243 Sentence denotes The pH dependence is strongest for PC1, SPF3 and INF7*.
TextSentencer_T190 24244-24355 Sentence denotes The leakage observed with INF7* was approximately 15-times higher at pH 5 than at pH 7.5 (Table 3 and Fig. 2) .
TextSentencer_T191 24356-24439 Sentence denotes The difference in leakage activity at pH 5 and pH 7.5 for PC2 is 3-fold (Table 3) .
TextSentencer_T192 24440-24542 Sentence denotes Leakage for PC1 and SFP3 at pH 7.5 was negligible even at the highest concentrations tested (Fig. 2) .
TextSentencer_T193 24543-24607 Sentence denotes Similar results were obtained with calcein-loaded ELM liposomes.
TextSentencer_T194 24608-24849 Sentence denotes Since most fusion or lytic peptides of enveloped and non-enveloped viruses are presented as part of a protein, often in multimeric form (42) , we tested the effect of oligomeric presentation of the membrane-active peptides PC1, PC2, and PC4.
TextSentencer_T195 24850-25032 Sentence denotes Using SPDP chemistry, peptides were coupled to poly (L-Lysine) (pLLys) (Mw = 30∼70 kDa) via a disulfide bond from their Cterminal cysteines to modified ε-amino groups of the lysines.
TextSentencer_T196 25033-25104 Sentence denotes With this procedure 16.6+/-1.7% of all lysines were carrying a peptide.
TextSentencer_T197 25105-25190 Sentence denotes Conjugates were then incubated with POPC liposomes in the presence or absence of DTT.
TextSentencer_T198 25191-25354 Sentence denotes In other words, peptides were either released from pLLys by reduction of the disulfide bridge in the presence of DTT, or remained conjugated in the absence of DTT.
TextSentencer_T199 25355-25521 Sentence denotes Analysis in the leakage assay showed that the conjugated form of PC1 was significantly more lytic than the reduced free peptide form (Fig. 3) at both pH 5 and pH 7.5.
TextSentencer_T200 25522-25680 Sentence denotes Despite the same trend, this effect was less pronounced for PC2-pLLys conjugates, and no activity could be observed for PC4-pLLys conjugates (data not shown).
TextSentencer_T201 25681-26024 Sentence denotes The reason for this behavior of PC2 and PC4 is not known at present, but the formation of aggregates might play a role in the case of PC4; PC2, on the other hand, is negatively charged and it is reasonable to assume that the positively charged pLLys may influence its leakage activity by neutralizing these charges and modulating the local pH.
TextSentencer_T202 26025-26117 Sentence denotes Poly (L-Lysine) alone does not result in leakage of calcein from POPC LUVs (data not shown).
TextSentencer_T203 26118-26360 Sentence denotes When studying the peptide sequences (Table 1) for their amino acid compositions two trends are immediately apparent: the great number of prolines and the differences in amino acid composition reflective of the MLV lipid composition (Fig. 4) .
TextSentencer_T204 26361-26581 Sentence denotes Those peptides arising from screens against acidic ELM MLVs show a particularly high preponderance of histidines and a concurrent paucity of aromatic and hydrophobic amino acid residues, with the exception of methionine.
TextSentencer_T205 26582-26697 Sentence denotes The sequences arising from phage screened against electroneutral POPC MLVs, on the other hand, are highly aromatic.
TextSentencer_T206 26698-26940 Sentence denotes To understand more quantitatively the peptide profile of the POPC screen, which yielded the membrane-active peptides, the frequency of amino acid occurrence was compared against its counterpart-frequencies in the parent Ph.D-12 library (Frig.
TextSentencer_T207 26941-26944 Sentence denotes 4).
TextSentencer_T208 26945-27114 Sentence denotes This analysis was weighted for peptide occurrence, i.e. the amino acid composition of each peptide sequence was multiplied with the frequency of occurrence of the clone.
TextSentencer_T209 27115-27311 Sentence denotes In the POPC MLV screen, from which the active peptides were identified, the aromatic amino acids are highly enriched (Tyr 1.6 x, Phe 2.6 x, Trp 4.8x enrichment above the parent library frequency).
TextSentencer_T210 27312-27419 Sentence denotes Not surprisingly, all titratable amino acids were enriched (aspartate 1.5x; glutamate 1.6x histidine 1.8x).
TextSentencer_T211 27420-27700 Sentence denotes When comparing the individual sequences from the membrane active peptides PC1, PC2 and PC4 to the non-active peptides PC3, PC5 and PC6, discovered in the screen using POPC MLVs, it is striking that none of the active peptides have either an arginine or a lysine in their sequence.
TextSentencer_T212 27701-27845 Sentence denotes In contrast, tryptophan is highly enriched in the sequence of two of the three active peptides but completely absent from the inactive peptides.
TextSentencer_T213 27846-27938 Sentence denotes Among the active peptides, the sequences are quite distinct in their amino acid composition.
TextSentencer_T214 27939-28097 Sentence denotes The sequence of PC2 is dominated by aliphatics (33% not counting the C-terminal Cysteine) in addition to a partially aliphatic methionine and a phenylalanine.
TextSentencer_T215 28098-28246 Sentence denotes These hydrophobic amino acids and the two acidic glutamates arranged in a periodic manner are features reminiscent of helical viral fusion peptides.
TextSentencer_T216 28247-28424 Sentence denotes PC1, on the other hand, has three titratable amino acids and has 2.8 x higher contents of aromatic amino acids and a 7.4 x higher contents of tryptophan than the parent library.
TextSentencer_T217 28425-28477 Sentence denotes The most intriguing sequence is the sequence of PC4.
TextSentencer_T218 28478-28584 Sentence denotes This peptide displays strongly pH-dependent lytic activity despite the lack of any titratable amino acids.
TextSentencer_T219 28585-28677 Sentence denotes Furthermore, all of its amino acids are overrepresented when compared to the parent library.
TextSentencer_T220 28678-28748 Sentence denotes Strikingly, tryptophan occurs 11.1 x more often than by random chance.
TextSentencer_T221 28749-28891 Sentence denotes It is tempting to speculate that the sequence diversity of the active peptides will be reflected in different mechanisms of membrane activity.
TextSentencer_T222 28892-29047 Sentence denotes In particular PC4, lacking any titratable groups, promises to provide novel insights in how peptides can induce pH-dependent permeabilization of liposomes.
TextSentencer_T223 29048-29128 Sentence denotes Secondary structures are known to be important in peptide-membrane interactions.
TextSentencer_T224 29129-29203 Sentence denotes Amphipathic helices are seen in many lytic (43) and fusion peptides (12) .
TextSentencer_T225 29204-29339 Sentence denotes Conversely, beta sheets can act on membranes (44) as beta aggregates (45, 46) , or they can insert into membrane as beta barrels (47) .
TextSentencer_T226 29340-29424 Sentence denotes We therefore analyzed the secondary structure of our peptides by circular dichroism.
TextSentencer_T227 29425-29556 Sentence denotes We first chose TFE as a co-solvent because it serves as a model for the hydrophobic environment of the aliphatic core of membranes.
TextSentencer_T228 29557-29846 Sentence denotes In 50% TFE and in the concentration range tested (50∼400 μM), SFP3 -a peptide designed to maximize its helical content and hydrophobic moment (41) -shows an α-helical spectra with minima at 208 nm and 222 nm and small contributions from random coil or possibly a 3 10 helix (48) (Fig. 5) .
TextSentencer_T229 29847-29959 Sentence denotes Two of our peptides, PC2, and to a lesser extent PC4 also show profiles consistent with α-helical conformations.
TextSentencer_T230 29960-30026 Sentence denotes The above structures are present between 25-55°C (data not shown).
TextSentencer_T231 30027-30161 Sentence denotes The partially helical conformation PC4 suggested by its CD spectrum is surprising given the preponderance of prolines and tryptophans.
TextSentencer_T232 30162-30251 Sentence denotes CD spectra of peptides, however, can be greatly influenced by aromatic amino acids (49) .
TextSentencer_T233 30252-30334 Sentence denotes Hence, it is possible that this apparent partially helical profile is artifactual.
TextSentencer_T234 30335-30448 Sentence denotes While PC1 shows pH-dependent lytic activity, notable structures were not observed under these solvent conditions.
TextSentencer_T235 30449-30514 Sentence denotes None of the other peptides showed any structure (data not shown).
TextSentencer_T236 30515-30797 Sentence denotes To study the effect of pH and membranes on the structure of our peptides, we next determined the secondary structure of the three active peptides and the positive control SFP3 in aqueous buffered solutions of pH 5 and pH 7.5, both in the presence and absence of liposomes (Fig. 6) .
TextSentencer_T237 30798-30918 Sentence denotes The signal-to-noise ratio could not be optimized to obtain spectra for PC1 and PC4 under the range of conditions tested.
TextSentencer_T238 30919-31069 Sentence denotes PC2 showed a more prominent alpha-helical conformation at pH 5 than at pH 7.5 where the spectra suggest a stronger influence of a typical random coil.
TextSentencer_T239 31070-31192 Sentence denotes Upon addition of POPC SUVs, the characteristic alpha-helical minima at 222 nm became more prominent, at both pH 5 and 7.5.
TextSentencer_T240 31193-31298 Sentence denotes SFP3 showed a similar pH-dependence with peptide alone: deeper 222 nm well minima at pH 5 than at pH 7.5.
TextSentencer_T241 31299-31452 Sentence denotes In contrast to PC2, the addition of liposomes at pH 7.5 did not result in increased helicity for SFP3 as reflected in calcein leakage results (Table 3) .
TextSentencer_T242 31453-31545 Sentence denotes Only in the presence of liposomes did the 222 nm well minima become more pronounced at pH 5.
TextSentencer_T243 31546-31723 Sentence denotes These trends of SFP3 are consistent with studies of the parent peptide GALA in presence of lipids (41) and with its sister peptide SFP1 in aqueous solution without lipids (40) .
TextSentencer_T244 31724-31944 Sentence denotes Taken together with results from Table 3 , the apparent rank order of lytic potencies does not seem to correlate with helicity, suggesting that additional parameters are influencing the lytic potential of these peptides.
TextSentencer_T245 31945-32164 Sentence denotes Nevertheless, the fact that both the lowering of pH as well as the addition of lipid membranes increase the helical profile of PC2 suggests a role of this structure in the pH-dependent membrane activity of this peptide.
TextSentencer_T246 32165-32377 Sentence denotes Future experiments, including additional spectroscopic methods and mutational analyses, will be needed to unravel the mechanisms of pH-dependent membrane activity of the diverse peptides discovered in this study.
TextSentencer_T247 32378-32571 Sentence denotes In this paper, we set out to discover peptides that disturb membranes in a pH-dependent manner by screening an M13 12-mer filamentous phage library, Ph.D.-12, for pH-dependent liposome binding.
TextSentencer_T248 32572-32735 Sentence denotes Two different liposome types were used: one that represents the aliphatic lipid bilayer core, POPC MLVs, and the second mimicking the endosomal membrane, ELM MLVs.
TextSentencer_T249 32736-32980 Sentence denotes In this context, we have identified 19 distinct peptide sequences, of which three peptides PC1, PC2, and PC4, are able to induce pH-dependent calcein leakage to a similar extent as that of the positive control peptides SFP3 and INF7* (Fig. 3) .
TextSentencer_T250 32981-33191 Sentence denotes In contrast to peptides identified from phage screens against receptors or antibodies (50), BLAST analysis showed that our panel of active peptides do not share significant homology with any proteins described.
TextSentencer_T251 33192-33271 Sentence denotes Instead, our peptides have varied sequences without an obvious consensus motif.
TextSentencer_T252 33272-33466 Sentence denotes This lack of a consensus sequence is unsurprising, since secondary (12) and multi-dimensional (44) homologies seem to be more important in defining membrane activity than their primary sequence.
TextSentencer_T253 33467-33743 Sentence denotes On the amino acid level, compositions of the discovered peptides reflect the nature of the lipids against which the screen has been conducted, aromatic groups against isoelectric POPC liposomes, and basic and less hydrophobic residues against negatively charged ELM liposomes.
TextSentencer_T254 33744-33836 Sentence denotes We have also enriched for histidines, a titratable amino acid in the pH-range of the screen.
TextSentencer_T255 33837-33951 Sentence denotes Against our expectations, aliphatic amino acids (with the exception of PC2) have not been enriched in our screens.
TextSentencer_T256 33952-34022 Sentence denotes Aromatics, on the other hand, were highly enriched in our POPC screen.
TextSentencer_T257 34023-34118 Sentence denotes Two of the membrane-active peptides -namely PC1 and PC4 -have a striking number of tryptophans.
TextSentencer_T258 34119-34310 Sentence denotes In contrast, trypthophan occurred only once in the 13 sequences discovered in the ELM screen and was completely absent from all the inactive peptides isolated from the POPC screen (Table 1) .
TextSentencer_T259 34311-34510 Sentence denotes When peptide activity was determined in vitro using the calcein leakage assay (26), we found that PC1, PC2 and PC4 were active both on electroneutral POPC as well as negatively charged ELM liposomes.
TextSentencer_T260 34511-34756 Sentence denotes These results imply that the calcein leakage activity of peptides is not dependent on ionic interaction with the lipid headgroups, but is instead governed by the ability of the peptides to interact with the hydrophobic core of the lipid bilayer.
TextSentencer_T261 34757-34896 Sentence denotes For PC1 and PC4, it is likely that the multiple tryptophans play an important role in this putative interfacial insertion into the bilayer.
TextSentencer_T262 34897-35091 Sentence denotes According to the Wimley-White interfacial hydrophobicity scale (51), aromatic and hydrophobic aliphatic residues (with the exception of valine) favor partitioning into a lipid bilayer (Fig. 4) .
TextSentencer_T263 35092-35299 Sentence denotes Tryptophan not only favors partitioning into bilayers more than aliphatics, but it is -unlike, for example, leucine -energetically undesirable to be located in the center of a transmembrane domain (16, 52) .
TextSentencer_T264 35300-35448 Sentence denotes The enrichment of tryptophans in the membrane-proximal domain has been implicated in the membrane fusogenic activity of viral fusion proteins (53) .
TextSentencer_T265 35449-35629 Sentence denotes The reason for the partial bilayer insertion of PC2, on the other hand, probably lies in its tendency to form an amphipathic helix as seen in classical viral fusion peptides (54) .
TextSentencer_T266 35630-35790 Sentence denotes In a helical conformation, the acidic glutamates and the hydrophobic amino acids (leucine, isoleucine and methionine) of PC2 lie on opposite faces of the helix.
TextSentencer_T267 35791-35935 Sentence denotes Another factor that can influence both membrane activity of peptides as well as the angle of peptide insertion into membranes is peptide length.
TextSentencer_T268 35936-36031 Sentence denotes Peptide length is often cited as a reason for diminished membrane activity of shorter peptides.
TextSentencer_T269 36032-36290 Sentence denotes While there are 6-mers (55) that have been shown to be membrane-active and 8 amino acid peptides are reported to interact stably with membranes (56), the membrane-active peptides studied in most detail are helices of a length of approximately 20 amino acids.
TextSentencer_T270 36291-36392 Sentence denotes One particularly well-studied peptide is the Nterminus of the influenza fusion protein hemagglutinin.
TextSentencer_T271 36393-36569 Sentence denotes In this model, using a host-guest system, increased peptide length increases both the depth and angle of membrane insertion as well contents leakage from red blood cells (57) .
TextSentencer_T272 36570-36835 Sentence denotes Given this information, it is remarkable that the 13 amino-acid-long peptides in our study enable contents leakage to a similar extent as the 24 amino-acid-long influenza fusion peptide analog INF7* (Table 3) , which has been optimized for its lytic activity (28) .
TextSentencer_T273 36836-36995 Sentence denotes Together these results suggest that the propensity of the membrane-active peptides to insert shallowly into the membrane is important for their lytic activity.
TextSentencer_T274 36996-37096 Sentence denotes The ability to penetrate deep into the hydrophobic core, on the other hand, may be less significant.
TextSentencer_T275 37097-37251 Sentence denotes The data are indeed consistent with a model in which a shallow interaction with the hydrophobic core of a bilayer is sufficient to induce content leakage.
TextSentencer_T276 37252-37388 Sentence denotes Most membrane-interacting synthetic peptides are more active as dimers (58, 59) and oligomers (60) or when anchored to a membrane (40) .
TextSentencer_T277 37389-37493 Sentence denotes This principle also holds true for two of our leakage active peptides, PC2 and especially PC1 (Fig. 3) .
TextSentencer_T278 37494-37708 Sentence denotes PC1 conjugated to poly(L-Lysine) by disulfide bonds, a multimeric presentation, is considerably more active in the calcein leakage assay than when the peptide was released from the oligomer in presence of 1 mM DTT.
TextSentencer_T279 37709-37843 Sentence denotes As stated in the introduction, membrane binding is obligatory, though not necessarily sufficient, for a peptide to be membrane-active.
TextSentencer_T280 37844-37967 Sentence denotes True to this prediction, peptides identified in our screen that lost the ability to bind stably to membranes are not lytic.
TextSentencer_T281 37968-38174 Sentence denotes The fact that in the context of the phage the peptides are both C-terminally anchored to a protein as well as being displayed in a pentameric form likely influences their ability to interact with membranes.
TextSentencer_T282 38175-38430 Sentence denotes Especially, because oligomeric presentation of the peptides almost certainly increases avidity, it is not surprising that some peptides -PC3, PC5 and PC6 as well as ELM1, ELM2 and ELM3 -lost the ability to bind stably to liposomes in their monomeric form.
TextSentencer_T283 38431-38472 Sentence denotes PC1 binds to both POPC and ELM liposomes.
TextSentencer_T284 38473-38696 Sentence denotes This is not unexpected since, as stated above, the interaction of PC1 with membranes is less likely to be influenced by the surface charge, but more likely by insertion of the peptide into the aliphatic core of the bilayer.
TextSentencer_T285 38697-38783 Sentence denotes Interestingly, the membrane-active peptides PC1, PC2 and PC4 bind to MLVs at both pHs.
TextSentencer_T286 38784-38932 Sentence denotes This appears to conflict both with the phage screen, which was based on the elution of the phages at pH 7.5, as well as the calcein leakage results.
TextSentencer_T287 38933-39083 Sentence denotes The simplest explanation for this behavior lies in the different natures of the liposome-binding assay and the phage screen and calcein leakage assay.
TextSentencer_T288 39084-39164 Sentence denotes In the phage screen, the eluted phages are amplified in each round of screening.
TextSentencer_T289 39165-39330 Sentence denotes Even if only a small amount of bound phage is eluted at pH 7.5, the bulk of the phage remaining bound to MLVs, it will be amplified and thus recovered in the screen.
TextSentencer_T290 39331-39419 Sentence denotes The liposome-binding assay, on the other hand, measures binding of the bulk of peptides.
TextSentencer_T291 39420-39547 Sentence denotes Despite the pH-independent binding of the peptides to MLVs, the lytic activity of all active peptides is strongly pH-dependent.
TextSentencer_T292 39548-39703 Sentence denotes Apparently, the lytic activity of our peptides is influenced by additional factors -such as pH-dependent structural changes -not just binding to liposomes.
TextSentencer_T293 39704-39867 Sentence denotes Alternatively, the reason for the difference between binding and lytic activity might lie in the different peptide and lipid concentrations used in the two assays.
TextSentencer_T294 39868-40043 Sentence denotes Because of the detection limit in the liposome binding assay, both the lipid and the peptide concentrations were substantially higher in the binding than in the leakage assay.
TextSentencer_T295 40044-40167 Sentence denotes The secondary structure of peptides has been instrumental in further understanding the interaction peptides with membranes.
TextSentencer_T296 40168-40254 Sentence denotes Many of the membrane-interacting components of viruses form amphipathic helices (12) .
TextSentencer_T297 40255-40552 Sentence denotes In line with these observations, we find that two of our peptides, PC2 and PC4, despite their short length, show helical propensities in 50% TFE (Fig. 5) ; although taking into account PC4′s highly aromatic amino acid composition, the "helical" structure must be confirmed by other methods (49) .
TextSentencer_T298 40553-40765 Sentence denotes The helicity of PC2 in aqueous solution shows a pH-and lipid-dependent increase, reminiscent of the conformational change and the resulting deeper membrane insertion of influenza fusion domain in acidic pH (61) .
TextSentencer_T299 40766-40882 Sentence denotes Thus, a model in which this change in helicity plays a role in the lytic activity of PC2 is particularly attractive.
TextSentencer_T300 40883-41007 Sentence denotes The fact that PC4 tends to aggregate in solution might also play an important role in the leakage potential of this peptide.
TextSentencer_T301 41008-41143 Sentence denotes Interestingly, aggregation of PC4 in solution was not strongly dependent on pH, in contrast to the pH-dependent leakage induced by PC4.
TextSentencer_T302 41144-41255 Sentence denotes This disparity suggests that differences exist in peptide organization in the absence or presence of membranes.
TextSentencer_T303 41256-41425 Sentence denotes These results are consistent with other studies showing that peptide assembly on membrane templates and aggregation has an important role in membrane activity (57, 59) .
TextSentencer_T304 41426-41542 Sentence denotes PC4 is of further interest because it possesses pH-dependent lytic activity in the absence of any titratable groups.
TextSentencer_T305 41543-41624 Sentence denotes One possible reason for the pH-dependency of PC4 is the presence of two prolines.
TextSentencer_T306 41625-41706 Sentence denotes Prolines can confer pH sensitivity by isomerizing in a pH-dependent manner (62) .
TextSentencer_T307 41707-41965 Sentence denotes The possible importance of prolines for the pH-dependent lytic activity of PC4 is highlighted by the fact that the probability of the double prolines at position 9 and 10 to occur by random chance in our screen is less than 10 -7 (see Materials and Methods).
TextSentencer_T308 41966-42103 Sentence denotes This indicates that these prolines are a result of the selective pressure in this screen for pH-dependent phage binding to the membranes.
TextSentencer_T309 42104-42342 Sentence denotes These findings resonate with the fact that centrally located prolines (63) , and their ability to change between secondary structures (55, 64, 65) , play an integral role in pH-dependent membrane destabilization induced by other peptides.
TextSentencer_T310 42343-42465 Sentence denotes Our work demonstrates that peptides that are membrane-active in a pH-dependent fashion can be identified by phage-display.
TextSentencer_T311 42466-42578 Sentence denotes The study also demonstrates that the choice of lipids used in the screen is important in selecting for activity.
TextSentencer_T312 42579-42755 Sentence denotes When the screen is performed with electroneutral PC liposomes that mimic the aliphatic core, 50% of the sequences discovered on the screen produced pH-dependent lytic peptides.
TextSentencer_T313 42756-43016 Sentence denotes Furthermore, membrane-active peptides -as opposed to their inactive counterparts -contain a remarkably high content of tryptophan, reminiscent of antimicrobial peptides (66), suggesting a crucial role of this amino acid in the lytic behavior of these peptides.
TextSentencer_T314 43017-43222 Sentence denotes Future studies aimed at deciphering the mechanism of membrane activity will help us to put these newly identified peptides into the context of both naturally occurring, as well as synthetic lytic peptides.
TextSentencer_T315 43223-43468 Sentence denotes The present approach may also open new avenues to gain a deeper understanding of peptide-lipid interactions and their role in cell biological and pathological processes, with the potential of yielding new strategies for therapeutic applications.
TextSentencer_T316 43469-43535 Sentence denotes Refer to Web version on PubMed Central for supplementary material.
TextSentencer_T317 43536-43611 Sentence denotes The authors would like to acknowledge the expert technical assistance of C.
TextSentencer_T318 43612-43665 Sentence denotes Cruickshank during the initial phase of this project.
TextSentencer_T319 43666-43696 Sentence denotes We would also like to thank J.
TextSentencer_T320 43697-43708 Sentence denotes Carlson, A.
TextSentencer_T321 43709-43865 Sentence denotes Kentsis and the Department of Structural Biology at Mount Sinai School of Medicine for their assistance in the use of the JASCO J-810 CD spectrometer and K.
TextSentencer_T322 43866-43874 Sentence denotes Senn, L.
TextSentencer_T323 43875-43885 Sentence denotes Gigout, R.
TextSentencer_T324 43886-43897 Sentence denotes Fransis, M.
TextSentencer_T325 43898-43908 Sentence denotes Linden, A.
TextSentencer_T326 43909-43923 Sentence denotes Thomas, and R.
TextSentencer_T327 43924-43960 Sentence denotes Brasseur for insightful discussions.
TextSentencer_T328 43961-44011 Sentence denotes A typical calcein release assay with PC1 is shown.
TextSentencer_T329 44012-44179 Sentence denotes Peptide was incubated at pH 5 and pH 7.5 in the presence of calcein-loaded phosphatidylcholine large unilamellar vesicles approximately 100 nm in diameter (POPC LUVs).
TextSentencer_T330 44180-44220 Sentence denotes PC1 and lipid concentrations were 25 μm.
TextSentencer_T331 44221-44257 Sentence denotes The incubation temperature was 37°C.
TextSentencer_T332 44258-44343 Sentence denotes Leakage was followed by measuring the increase in calcein fluorescence at 485/537 nm.
TextSentencer_T333 44344-44434 Sentence denotes At 30 minutes (arrowhead), Triton X-100 (TX100)was added to a final concentration of 0.2%.
TextSentencer_T334 44435-44616 Sentence denotes Percent leakage was calculated by setting fluorescence of liposomes in buffer at time = 0 min. as 0% leakage and fluorescence after complete lysis by TX100 addition as 100% leakage.
TextSentencer_T335 44617-44625 Sentence denotes Symbols:
TextSentencer_T336 44626-44697 Sentence denotes 25 μM PC1 at pH 5 (filled circles); 25 μM PC1 at pH 7.5 (open circles).
TextSentencer_T337 44698-44908 Sentence denotes Leakage studies were conducted as described for Figure 1 at various peptide concentrations (0 to 75 μM, except for PC4, for which the concentration range was 0 to 8.3 μM due to peptide aggregation in solution).
TextSentencer_T338 44909-45081 Sentence denotes Extent of leakage after 30 minutes of incubation with peptide was plotted against peptide concentration as an average of three independent experiments +/standard deviation.
TextSentencer_T339 45082-45137 Sentence denotes Symbols: pH 5 (filled circles); pH 7.5 (empty circles).
TextSentencer_T340 45138-45188 Sentence denotes Fifty percent leakage is denoted by a dotted line.
TextSentencer_T341 45189-45304 Sentence denotes Calcein leakage in the presence of PC1-poly(L-Lysine) (pLLys) at 25 μM PC1 was followed as described for Figure 1 .
TextSentencer_T342 45305-45456 Sentence denotes Peptides were coupled via their cysteine residues to the ε-amino groups of 46 kDa pLLys at 16.6+/-1.7%, as described in (24) and Materials and Methods.
TextSentencer_T343 45457-45465 Sentence denotes Symbols:
TextSentencer_T344 45466-45679 Sentence denotes Conjugated peptides at pH 5 (closed circles); free peptides (i.e., released from pLLys with 1 mM DTT) at pH 5 (closed squares); conjugated peptides at pH 7.5 (open circles); free peptides at pH 7.5 (open squares).
TextSentencer_T345 45680-45725 Sentence denotes Arrowhead denotes the time of TX100 addition.
TextSentencer_T346 45726-45857 Sentence denotes The frequency of amino acids in the peptides discovered from the screen was normalized against that of the parent Ph.D.-12 library.
TextSentencer_T347 45858-46044 Sentence denotes The parent library's amino acid frequency has been determined previously and was calculated from the sequences of 100 randomly selected phage clones (see Materials and Methods and (39)).
TextSentencer_T348 46045-46167 Sentence denotes The amino acids are ordered according to the residue's propensity of partitioning into either water or liposomal bilayers.
TextSentencer_T349 46168-46280 Sentence denotes This rank order is depicted according to the Wimley-White water/bilayer whole residue hydrophobicity scale (51).
TextSentencer_T350 46281-46443 Sentence denotes Wimley-White obtained this scale by experimentally determining the free energy of transfer (kcal·mol -1 ) of an amino acid residue from water into a POPC bilayer.
TextSentencer_T351 46444-46561 Sentence denotes Each peptide sequence that was isolated more than once was counted as a distinct peptide for each of the occurrences.
TextSentencer_T352 46562-46718 Sentence denotes Circular Dichroism spectra were taken of peptides dissolved in TFE and measured at 50% TFE in water, 1 mM TCEP at 25+/-2oC on a JASCO J-810 CD spectrometer.
TextSentencer_T353 46719-46838 Sentence denotes The spectra represented in molar ellipticity units did not change within the peptide concentrations range of 50∼400 μM.
TextSentencer_T354 46839-46928 Sentence denotes Peptides were dissolved into 0.1% ammonia stock solution, from which dilutions were made.
TextSentencer_T355 46929-47056 Sentence denotes Final CD buffer was 0.01% ammonia, 2 mM citrate, 2 mM borate, 2 mM potassium phosphate, with 10 mM sodium chloride, 10 mM TCEP.
TextSentencer_T356 47057-47138 Sentence denotes The buffering components were used to adjust the solutions to the appropriate pH.
TextSentencer_T357 47139-47208 Sentence denotes Measurements were taken on a JASCO J-810 CD spectrometer at 25+/-2°C.
TextSentencer_T358 47209-47302 Sentence denotes Addition of lipids did not cause a disturbance in the baseline measurements (data not shown).
TextSentencer_T359 47303-47457 Sentence denotes Symbols: pH 5 (closed inverted triangles); pH 7.5 (open inverted triangles); pH 5 with POPC SUVs (closed diamonds); pH 7.5 with POPC SUVs (open diamonds).
TextSentencer_T360 47458-47639 Sentence denotes Peptide-to-lipid ratios were less than 0.6. * * + + a Peptide binding was assessed by incubating 250 μM peptide with 2.5 mM MLVs in either pH 5 or 7.5 buffer for 30 minutes at 37°C.
TextSentencer_T361 47640-47976 Sentence denotes After pelleting the MLVs by centrifugation, both the supernatant and pellet were analyzed for the presence of peptides by thin layer chromatography (TLC) and visualization by ninhydrin staining. *The amino lipids in ELM MLVs could not be resolved from PC2 and PC4 making the analysis of binding of these peptides to ELM MLVs impossible.