From the Departments of Pathology and
** Microbiology & Molecular Genetics, University of
California College of Medicine, Irvine, California 92697-4800 and
the
Department of Chemistry, Ben-Gurion University of the Negev,
Beersheva, Israel 84105
Received for publication, November 27, 2002, and in revised form, January 29, 2003
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ABSTRACT |
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The bactericidal activity of mouse
Antimicrobial peptides have been identified as components of
innate immunity in every living organism investigated (1), and the
mammalian Human and rabbit neutrophil In this work, the newly developed lipid/polydiacetylene (PDA)
colorimetric vesicle assay combined with fluorescence and CD spectroscopies have been applied to characterizing Crp4 interaction and
permeation of membranes. The lipid/PDA system consists of mixed
vesicles composed of PDA interspersed with natural lipids, and the
vesicles undergo blue-red transitions when induced by a variety of
biological molecules that interact with the vesicular lipid components.
The polymeric PDA matrix, which forms the scaffold of the mixed
vesicles, accommodates the incorporation of varied lipid components
while maintaining an overall sensitivity to membrane properties and
processes. Analyses of PDA-based mixed vesicles have proven useful in
studies of varied membrane-related processes, including
peptide-membrane interactions (16, 17), membrane permeation by
penetration enhancers (18), and biological recognition events occurring
at membrane interfaces (19). In particular, this colorimetric assay has
provided insights into mechanisms of membrane permeation, including the
degree of interfacial disruption by peptides, the depth of peptide
penetration into the lipid bilayer, and changes in membrane fluidity in
response to peptide exposure (16,
17).2 Crp4 was chosen for
this study, because this secreted peptide functions in the
extracellular compartment (20, 21) and is the most bactericidal mouse
Paneth cell In the studies reported here, interactions between model membranes and
mouse Crp4 and its precursor have been analyzed spectroscopically and
by colorimetric measurements on lipid/PDA mixed vesicles, and those
interactions were correlated with the bactericidal activity of the
peptide. The mechanisms of Crp4 membrane binding and permeation and the
sensitivity of that binding to the composition of the lipid bilayer
were evaluated in relation to melittin, a classic amphipathic
Materials--
Phospholipids, including
dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol
(DMPG), and lipopolysacharides (LPS from Escherichia coli
055:B5) were purchased from Sigma-Aldrich). LPS was dialyzed
sequentially against 1 mM EDTA for 24 h and against distilled water to remove excess divalent cations, which can cause lipid/PDA vesicles to precipitate. The diacetylenic monomer,
10,12-tricosadiynoic acid, was purchased from GFS Chemicals (Powell,
OH), washed in chloroform, and filtered through a 0.45-µm filter
prior to use. For commercially obtained peptides, melittin was
purchased from Sigma-Aldrich, and the Crp1 proregion consisting of the
following primary structure: DPIQNTDEET KTEEQPGEDD QAVSVSFGDP EGTSLQEES was synthesized by Quality Controlled Biochemicals, Inc. (Hopkinton, MA). The properties of the synthetic prosegment have been reported previously (10).
Lipid Extraction from Bacteria--
E. coli strain
B/r H-266 was grown at 37 °C for 24 h in LB medium. The lipids
were extracted from E. coli by resuspending deposited
bacterial cells in 4 M NaCl and an equal volume of a 1:1
mixture of chloroform and methanol. After the mixture was shaken gently
for 1 h and refrigerated overnight, the chloroform and aqueous
phases were separated by centrifugation at 5000 × g
for 15 min, and the aqueous methanol solution was re-extracted with
chloroform. The combined chloroform extracts were concentrated by
solvent evaporation, and the residual lipid-containing fraction was
lyophilized. The lipids were stored at Preparation of Recombinant Cryptdin-4 Peptides--
Recombinant
Crp4, the G1W-Crp4 variant, and procryptdin-4 (pro-Crp4) were expressed
in E. coli as N-terminal His6-tagged fusion proteins. DNA coding for the Crp4 peptide, corresponding to nucleotides 182-274 of mouse Crp4 cDNA (27), was amplified and cloned in-frame with the N-terminal His6 in the
EcoRI/SalI sites of pET-28a (Novagen, Inc.,
Madison, WI). The Crp4 coding cDNA sequences were amplified using
forward primer (ER1-Met-C4-F), 5'-GCGCG AATTC ATCGA GGGAA GGATG GGT TTGTT ATGCT ATTGT, paired with reverse primer
(pMALCrp4-R), 5'-ATATA TGTCG ACTCA GCGAC AGCAG AGCGT GTACA ATAAA TG
(18). A Trp for Gly substitution was made at Crp4 N terminus by pairing the common reverse primer (pMALCrp4-R) with forward primer
(ER1-Met-G1W-F) 5'-ATATG AATTC ATGTGGTTGTTATGCTAT to
prepare G1W-Crp4. To prepare recombinant pro-Crp4, a Met-coding
trideoxynucleotide was incorporated 5' of codon 20 in the Crp4
precursor cDNA and cloned in pET-28a as above. For cloning
pro-Crp4, forward primer pETPCr4-F (5'-GCGCG AATTC
ATGGA TCCTA TCCAA AACAC A) was paired with reverse primer SLpMALCrp4R (5'-ATATA TGTCG ACTGT TCAGC GGCGG GGGCA GCAGT ACAA). The reactions were performed by incubating the reaction mixtures at
95 °C for 5 min, followed by successive cycles at 60 °C for 1 min, 72 °C for 1 min, and 94 °C for 1 min for 40 cycles. The underlined codon in each forward primer denotes a Met codon to incorporate a CNBr cleavage site immediately upstream of the designed peptide N terminus.
Recombinant proteins were expressed in E. coli
BL21(DE3)-CodonPlus-RIL cells (Stratagene Cloning Systems, Inc., La
Jolla, CA) transformed with appropriate cDNA constructs. The cells
were grown at 37 °C to A620 = 0.9 in
Terrific Broth medium consisting of 12 g of Bacto Tryptone (Becton
Dickenson Microbiological Systems, Inc., Sparks, MD), 24 g of
Bacto Yeast Extract (Becton Dickenson), 4 ml of glycerol, 900 ml of
H2O, 100 ml of sterile phosphate buffer consisting of 0.17 M KH2PO4, 0.72 M
K2HPO4, and 70 µg/ml kanamycin (28).
Expression of fusion proteins was induced with 0.1 mM isopropyl- Purification of Recombinant Crp4 and pro-Crp4
Proteins--
His-tagged Crp4 fusion proteins were purified using
nickel-nitrilotriacetic acid resin affinity chromatography by
incubating cell lysates with nickel-nitrilotriacetic acid resin
(Novagen, Madison, WI) at a ratio of 25:1 (v/v) in 6 M
guanidine HCl, 20 mM Tris-HCl (pH 8.1) for 4 h at
4 °C. Fusion proteins were eluted with 2 column volumes of 6 M guanidine HCl, 1 M imidazole, 20 mM Tris-HCl (pH 6.4) dialyzed against 5% acetic acid
(HOAc) in SpectraPor 3 dialysis membranes (Spectrum Laboratories, Inc., Rancho Dominguez, CA) and lyophilized. The Met residue introduced at
the Crp4, G1W-Crp4, and pro-Crp4 N termini was subjected to CNBr
cleavage by dissolving lyophilized His6 fusion proteins in 50% formic acid, addition of solid CNBr to 10 mg/ml final
concentration, and incubation of the mixtures for 8 h in darkness
at 25 °C. The cleavage reactions were terminated by addition of 10 vol of H2O, followed by freezing and lyophilization of the
peptide mixture. The cleaved fusion peptide mixtures were dissolved in
5% acetic acid and stored at 4 °C.
Recombinant proteins were purified to homogeneity using reverse phase
high performance liquid chromatography (RP-HPLC). After CNBr cleavage,
Crp4, G1W-Crp4, and pro-Crp4 peptides were separated from CNBr
fragments of the 36-amino acid His6 tag fusion partner by
C-4 RP-HPLC on a Vydac 214TP1010 column (Vydac, Hesperia, CA). For
Crp4, the samples were applied to C-4 columns in aqueous 0.1% trifluoroacetic acid, and the peptides were resolved with a 0-35% acetonitrile gradient developed over 55 min. All other peptides were
purified to homogeneity by analytical C-18 RP-HPLC on a Vydac 218TP54
column. Using the same mobile phase, recombinant proteins were resolved
with elution times ranging from 21 to 24 min using a 10-45%
acetonitrile gradient over 55 min. Protein fractions containing Crp4
were identified by acid-urea PAGE as described (14) to confirm
co-migration with natural Crp4 and pro-Crp4 and to evaluate the
homogeneity of the preparation. Peptide concentrations were quantified
by amino acid analysis (Waters Alliance, Bedford, MA) or UV absorption
at 280 nm based on the extinction coefficients of the individual
proteins. Molecular masses of purified peptides were determined using
matrix-assisted laser desorption ionization mode mass spectrometry
(Voyager-DE MALDI-TOF; PerkinElmer Life Sciences) in the UCI Biomedical
Protein and Mass Spectrometry Resource Facility.
Bactericidal Peptide Assays--
Recombinant peptides were
tested for microbicidal activity against E. coli ML35 and
the PhoP Vesicle Preparation--
Vesicles containing lipid components
and PDA (DMPG/DMPC/PDA, 1:1:3 ratio; LPS/DMPC/PDA, 0.2:2:3 ratio) were
prepared as follows. All of the lipid constituents were dissolved in
chloroform/ethanol (1:1), dried together in vacuo to
constant weight, and suspended in deionized water by probe sonication
at 70 °C for 2-3 min. The vesicle suspension was cooled to room
temperature, incubated overnight at 4 °C, and polymerized by
irradiation at 254 nm for 10-20 s, resulting in solutions having an
intense blue appearance.
Ultracentrifugation Binding Assay--
An ultracentrifugation
binding assay was carried out for evaluation of peptide affinities, or
partition coefficients, to the vesicles. First, a calibration graph
that correlated peptide concentration with the absorbance at 220 nm was
prepared and used to determine the concentration of soluble, unbound
peptide. Varying quantities of peptides were added to aqueous lipid/PDA
vesicle solutions containing 0.5 mM total lipid in 25 mM Tris-HCl (pH 8), and the solutions were incubated
briefly at ambient temperature to allow equilibration of bound and
unbound peptide species, followed by centrifugation at 30,000 rpm for
40 min in a SW-55 rotor to deposit vesicle-peptide aggregates. The
concentration of soluble, i.e. unbound, peptide in the
supernatant was determined by extrapolation from the calibration curve,
and the quantity of bound peptide was calculated as the difference from
the initial peptide concentration. The binding data results were
confirmed using the Lowry method for determination of soluble peptide
concentration (24).
UV-visible Measurements--
Spectral UV-visible measurements
were carried out to analyze and quantify the colorimetric transitions
undergone by the blue lipid/PDA vesicles. Peptides at concentrations
ranging from 0.2 to 20 µM were added to 60-µl vesicle
solutions consisting of 0.5 mM total lipid in 25 mM Tris-HCl (pH 8). Following addition of the peptides, the
solutions were diluted to 1 ml, and spectra were acquired at 28 °C
between 400 and 700 nm on a Jasco V-550 spectrophotometer (Jasco Corp.,
Tokyo), using a 1-cm optical path cell.
The extent of blue-to-red color transitions within the vesicle
solutions, the colorimetric response (%CR), was defined and calculated
as follows (19).
Fluorescence Measurements--
Changes in tryptophan intrinsic
emission were measured for 10 µM peptide solutions
titrated with vesicles. Fluorescence emission spectra were acquired at
28 °C on an FL920 spectrofluorimeter (Edinburgh Co., Edinburgh, UK),
using excitation at 280 nm and emission at 345 nm. Excitation and
emission slits were both 8 nm. Total sample volumes were 1 ml, and the
solutions were placed in a quartz cell having a 0.5-cm optical path
length. Light scattering from the vesicles was confirmed to account for
less than 5% of the emission intensity.
Circular Dichroism--
CD spectra were acquired on an Aviv
62A-DS Circular Dichroism Spectrometer (Aviv Inc., Lakewood, NJ). Four
scans were recorded between 190 and 250 nm with 1-nm acquisition steps.
A 0.2-mm optical path length was used. All of the vesicle solutions had
a total lipid concentration of 1 mM in 50 mM
Tris-HCl at pH 8. The peptide concentrations were 0.1 mM.
Properties and Bactericidal Activities of Recombinant Crp4 and
pro-Crp4 Peptides--
Consistent and efficient recombinant expression
of Crp4, G1W-Crp4, and pro-Crp4 (Fig.
1A) was obtained using the
pET-28 vector system. Recombinant His6-tagged fusion
proteins were purified by affinity chromatography from the bacterial
cell lysates ("Experimental Procedures"). After chemical cleavage
with CNBr, Crp4, G1W-Crp4, and pro-Crp4 were purified to homogeneity by
RP-HPLC ("Experimental Procedures"). Peptide homogeneity was
evaluated using analytical RP-HPLC (not shown) and acid-urea PAGE (Fig.
1B). All of the purified peptides were homogeneous by these
criteria, migrated as expected relative to native Crp4 and pro-Crp4
molecules (Fig. 1B),3 and the molecular masses
of individual recombinant peptides determined by MALDI-TOF mass
spectrometry matched the respective theoretical values exactly. Thus,
the purified recombinant peptides were homogeneous and biochemically
equivalent to the natural molecules.
The in vitro microbicidal activities of Crp4, G1W-Crp4, and
pro-Crp4 were evaluated against the defensin-sensitive
PhoP
Melittin was selected as a model peptide for comparisons of
bactericidal activity and membrane interactions with Crp4. The folded
conformations of Crp4 and melittin differ. Crp4 is a typical Colorimetric Analysis of Crp4-Membrane Interactions--
Previous
studies of neutrophil
To evaluate the membrane association properties of Crp4 more
completely, the relative peptide affinities of Crp4 and melittin for
the lipid/PDA mixed vesicles were determined. The relative binding of
melittin and Crp4 to vesicles was measured using an ultracentrifugation
binding assay ("Experimental Procedures"), recording the partition
coefficients of each peptide in the lipid/PDA vesicle environment (32).
The binding assays showed that the initial linear slopes of Crp4 and
melittin binding to vesicles in aqueous solutions are almost identical,
yielding partition coefficients of ~0.9 for DMPC/PDA vesicles (Fig.
4A) and 0.75 for DMPG/DMPC/PDA
vesicles (Fig. 4B). These results show that both peptides
have similar affinities for lipid/PDA vesicles of different
compositions (32). However, as evident from the plateaus corresponding
to the quantities of each peptide bound at saturation, melittin and
Crp4 differ extensively with respect to the maximal concentration of
peptide bound to these model membranes. For example, both vesicle
assemblies bound ~6 µM melittin at saturation (Fig. 4)
but a maximum of only 1 µM Crp4. These findings suggest
that the lower quantity of bound Crp4 results from incorporation of the
peptide at the lipid/water interface (Fig. 4). In contrast, melittin
adopts a helical amphipathic structure in hydrophobic membrane
environments (23) and inserts deeply into lipid bilayer assemblies (33,
34), leading to accumulation of higher concentrations of
membrane-associated melittin relative to levels of membrane-bound Crp4.
Crp4 and melittin exhibit different membrane association profiles as
well as differential sensitivities to phospholipid components of the
vesicles. Generally, the bactericidal activity of antimicrobial peptides is consistent with the ability to penetrate and disrupt membranes (14). Fig. 5 depicts curves
corresponding to the %CR induced by increasing quantities of bound
peptides, i.e. the extent of induced blue-red transitions
affected by the added peptides. Vesicles of three phospholipid
compositions were investigated: DMPC/PDA at a 2:3 molar ratio (Fig.
5A), DMPG/DMPC/PDA at a 1:1:3 molar ratio (Fig.
5B), and vesicles using total cell lipids extracted from
E. coli ("Experimental Procedures") at a 2:3 mass ratio
of total lipid to PDA (Fig. 5C). The results in Fig. 5 show
that %CR values correlate with the concentration of vesicle-bound
peptide after accounting for the partition coefficients determined by ultracentrifugation binding assays (Fig. 4). Thus, the curves reveal
that each peptide interacts with these membrane phospholipids differently, particularly with respect to the degree of penetration into the lipid layer.
Previously, the localization of peptide at the lipid bilayer surface
was shown to induce a greater increase in %CR as a function of the
quantity of bound peptide, and peptides that penetrate deeper into the
hydrophobic core of the membrane bilayer produce a lower rise in
chromatic shift (16). In principle, a direct relationship exists
between higher %CR and interfacial lipid binding, because the
mechanism of colorimetric transformation of the polymer assumes an
increased mobility of the pendant side chains, induced through
perturbations at the lipid/PDA vesicle surface (16, 17). In all three
lipid systems, Crp4 gave rise to steeper increases in %CR than
melittin at peptide concentrations
The interactions of Crp4 and melittin with the biomimetic membrane
system exhibit differential sensitivity to mixed vesicle lipid
composition (Fig. 6). The experiments
summarized in Fig. 6 were designed to investigate the specificity of
the peptides to different membrane models, i.e. bacterial
versus eukaryotic membranes. For example, the bilayer
interactions evident from the %CR curves induced by Crp4 diverge
markedly for vesicles of differing lipid compositions (Fig.
6A). In contrast, melittin interactions are almost
superimposable, regardless of vesicle composition used in these
experiments (Fig. 6B). The steepest %CR curve was induced
by Crp4 in LPS-containing DMPC/PDA vesicles (60%CR at 0.5 µM Crp4), but 0.5 µM Crp4 induced only
~40%CR in DMPC/PDA vesicles (Fig. 6A). Interestingly,
Crp4 inserted deeper into the lipid bilayer when the lipid/PDA matrix
included DMPG or E. coli total lipids as shown by the
respective 20 and 10%CR values in the presence of 0.5 µM
Crp4 (Fig. 6A). Also, the increase in %CR as a function of
Crp4 concentration was lower by almost one-half for DMPG or E. coli total lipid-containing vesicles. In contrast to these
pronounced effects of lipid composition upon Crp4 bilayer penetration,
interactions between melittin and vesicles were indistinguishable,
regardless of vesicular phospholipid content (Fig. 6B).
These results are likely to result from the high overall attraction of
melittin to negatively charged membrane surfaces (PDA head groups are
ionized with basic pH conditions). At peptide concentrations identical
to those for Crp4, melittin elicited respective 4-6-fold lower %CR
values for DMPC/PDA and DMPG/DMPC/PDA vesicles, indicating deeper
insertion by melittin.
Tryptophan Fluorescence Measurements--
The incorporation of
Crp4 into lipid/PDA vesicles was confirmed further by Trp fluorescence
measurements and comparisons with melittin. The intrinsic fluorescence
emission of the Trp indole ring is sensitive to the hydrophobicity of
the local environment of the side chain (35, 36). Because peptides
insert more deeply into the acyl core of the lipid bilayer, the
environment of the Trp residue becomes more hydrophobic (35), shifting
the fluorescence emission peak to higher wavelengths over the course of
peptide-membrane associations. To perform these studies, the G1W-Crp4
analog was prepared with a Trp for Gly substitution at the peptide N
terminus ("Experimental Procedures" and Fig. 1B).
G1W-Crp4 is bactericidal (Fig. 1C) and exhibits lipid
binding and colorimetric properties that are very similar to those of
Crp4 (data not shown), evidence that the G1W replacement at the Crp4 N
terminus does not alter the membrane disruptive properties of the
peptide substantially. The native melittin primary structure contains a
single Trp residue at position 19, which has been used extensively as a
fluorescent probe for investigating melittin-membrane interactions (35, 36).
The observed shifts in the Trp fluorescence peaks resulting from
G1W-Crp4 and melittin interactions with DMPC/PDA and DMPG/DMPC/PDA vesicles are summarized in Fig. 7. When
G1W-Crp4 interacts with DMPG/DMPC/PDA vesicles, the Trp fluorescence
emission signal shifted 15 nm, from 355 to 340 nm, at a lipid to
peptide ratio of 10:1 (Fig. 7, upper panel). A
smaller 10-nm fluorescence shift, from 352 to 342 nm, was
observed for DMPC/PDA vesicles (Fig. 7, upper panel). Consistent with the colorimetric data (Fig.
5B), G1W-Crp4 inserts more deeply into the lipid moiety of
the DMPG/DMPC/PDA assemblies relative to insertion into DMPC/PDA
vesicles (Fig. 7, upper panel). Also consistent with the
colorimetric measurements (Fig. 5B), the emission shifts
produced by melittin were similar whether interacting with
DMPG/DMPC/PDA or DMPC/PDA vesicles (Fig. 7, lower panel),
showing that melittin was incorporated into vesicles to the same extent
regardless of the lipid composition. As further evidence that melittin
inserts into the lipid bilayers more deeply than G1W-Crp4, melittin
shifted the Trp emission peak more than G1W-Crp4 regardless of the
phospholipid composition of the vesicles (Fig. 7, lower
panel).
Crp4 Precursor Does Not Perturb Lipid/PDA
Vesicles--
Consistent with its lack of bactericidal activity (Fig.
1C), pro-Crp4 did not interact with lipid/PDA model
membranes. Mouse cryptdins are processed from inactive pro-Crp
precursors by MMP-7-mediated proteolysis (10), which is essential for
the activation of bactericidal mature Prosegment Inhibits Crp4 Lipid/PDA Vesicle
Interactions--
Soluble, intact prosegments inhibit the in
vitro bactericidal activity of mature
Addition of the Crp1 proregion in trans exhibited similar
inhibitory effects on G1W-Crp4 interactions with membrane vesicles as
measured by shifts in Trp fluorescence emission. As shown in Fig.
8C, the prosegment blocked G1W-Crp4 incorporation into the lipid moieties, consistent with inhibition of %CR by the proregion (Fig. 8B). Compared with the 10-nm Trp emission peak shift
recorded for G1W-Crp4, a peak shift of only 1 nm was recorded for
G1W-Crp4 when preincubated with prosegment at 0.6 mol of G1W-Crp4/mol
of prosegment, concentrations that inhibit Crp4 bactericidal activity (10). Again, prosegment alone did not alter Trp fluorescence emission
(data not shown). This negligible Trp shift supports the conclusion
that G1W-Crp4 associations with the lipid bilayer are blocked or
minimized in the presence of equimolar or greater concentrations of
intact proregion. However, at Crp4 to proregion molar ratios of 4:1,
the ~8-nm shift in Trp fluorescence approached that of G1W-Crp4
alone. These findings support a model in which the proregion
association with Crp4, possibly through charge neutralization at
specific residue positions, inhibits peptide bactericidal activity by
interfering with peptide-membrane interactions.
Analyses of CD spectra were consistent with the interpretation of the
preceding membrane perturbation experiments and provided insights into
the structural properties of native Crp4, its inactive Crp4 precursor,
and Crp4 in the presence of inhibitory concentrations of Crp1
prosegment (Fig. 9). In aqueous solutions
of DMPC/PDA, native Crp4 exhibits a characteristic Prosegment proteolysis during MMP-7-mediated pro-Crp processing
results in activation of cryptdin bactericidal activity from inactive
proforms.3 The underlying biophysical effects of the
prosegment upon the Crp4 sequence, however, are not known. The results
of the colorimetric and spectroscopic analyses presented here support
the conclusion that the pro-Crp4 proregion maintains the Crp4 peptide
in an inactive state by inhibiting interactions between Crp4 and lipid
bilayers (Fig. 8). Consistent with this notion, similar molecular
mechanisms appear to operate when prosegment, added in
trans, prevents membrane association between vesicles and the
mature Crp4 peptide. Of course, caution should be exercised in
extrapolating from these in vitro studies to the factors
that may be associated with killing mechanisms in vivo.
Recent findings show that MMP-7 activation of pro-Crp4 is independent
of proteolysis at two sites within the proregion,3 but the
detailed molecular mechanisms by which MMP-7 cleavage eliminates the
inhibitory effects of the prosegment remain to be elucidated. The
experimental approaches described here appear to provide the level of
sensitivity and selectivity required for defining those mechanisms.
Crp4 membrane interactions are sensitive to the phospholipid
constituents of the lipid bilayer component in the PDA mixed vesicles.
These findings are consistent with the ability of rabbit The apparent interfacial lipid binding and perturbation of Crp4
in the biomimetic model membrane suggests that Crp4 associates and
permeates the membrane bilayer by mechanisms that are distinct from
those of melittin. Moreover, the bactericidal and membrane permeation
differences between Crp4 and melittin are most likely consequences of
the distinctly different structural and functional properties of the
two peptides. Comparisons of the colorimetric transitions induced in
lipid/PDA vesicles incorporating different lipid/PDA ratios (Fig. 3)
confirmed that the chromatic transformations correspond to specific
interactions between the peptides and the lipid components incorporated
within the PDA matrices. The colorimetric and Trp fluorescence
experiments are consistent with a model indicating that Crp4 undergoes
interfacial binding onto lipid bilayers with disruption of the bilayer
head group region. Furthermore, Crp4 interactions at the lipid/water
interface are more pronounced than those induced by melittin, which
inserts more deeply into the hydrophobic core of the vesicles. The
interfacial binding of Crp4, the absence of significant peptide
insertion into the bilayer, and the apparent dependence of
peptide-membrane interactions on vesicle lipid composition suggest that
Crp4 cytolytic mechanism(s) differ from those of linear amphipathic
peptides that adopt helical conformations to facilitate lipid bilayer
penetration. In particular, models such as the barrel-stave (39) or
carpet mechanism (40, 41) are not consistent with our results and not
adequate to explain the membrane permeation properties of Crp4.
Colorimetric analysis demonstrates that Crp4-membrane association,
characterized extensively in Figs. 3-7, is attenuated by the
prosegment and that this effect depends on the prosegment to Crp4 ratio
(Fig. 8). Measurements of Trp fluorescence spectra independently showed
that prosegment also inhibited G1W-Crp4 insertion into lipid bilayers
when the peptide was incubated with prosegment before mixing with
vesicles (Fig. 8). Furthermore, CD experiments provided evidence that
the -defensins (cryptdins) requires proteolytic activation of inactive
precursors by matrix metalloproteinase-7 (matrilysin, EC 3.4.24.23,
MMP-7a). To investigate mechanisms of cryptdin-4
(Crp4) peptide interactions with membrane bilayers and to determine
whether MMP-7-mediated proteolysis activates the membrane disruptive
activity of Crp4, associations of Crp4 and melittin with biomimetic
lipid/polydiacetylene chromatic vesicles were characterized. The
peptides differ in their sensitivity to vesicle lipid composition and
their depth of bilayer penetration. Crp4 undergoes strong interfacial
binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies
showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli
phospholipids promote stronger interfacial binding; melittin-membrane
interactions were independent of either variable. In contrast to the
membrane disruptive activity of Crp4, pro-Crp4 did not perturb
vesicular membranes, consistent with the lack of bactericidal activity
of the precursor, and incubation of Crp4 with prosegment in
trans blocked Crp4 and G1W-Crp4 membrane interactions at
concentrations that inhibit Crp4 bactericidal activity. CD
measurements showed that Crp4 has an expected
-sheet structure that
is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with
prosegment, indicating that the
-sheet signal is attenuated by
proregion interactions or possibly disrupted by the prosegment.
Collectively, the results suggest that the prosegment inhibits Crp4
bactericidal activity by blocking peptide-mediated perturbation of
target cell membranes, a constraint that is relieved when MMP-7 cleaves
the prosegment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-defensins were among the first antimicrobial peptide
families to be recognized and characterized (2, 3).
-Defensins are
major constituents of both azurophilic granules in mammalian phagocytic
leukocytes and secretory granules of mammalian Paneth cells, where they
are termed cryptdins (Crps)1
in mice (4, 5). In contrast to
-defensins in cells of myeloid
origin, which provide a nonoxidative means for killing microorganisms
after phagocytosis (6), Paneth cell
-defensins are secreted to
function in the extracellular compartment (7, 8). These cationic
peptides with molecular masses of 3-4 kDa contain six cysteine
residues that form a distinctive tridisulfide array to produce an
amphipathic peptide, a feature that is essential for its microbicidal
activity (6). In mouse Paneth cells, the production of mature
bactericidal
-defensins requires proteolytic activation by matrix
metalloproteinase-7 (9), a process that precedes secretion (10).
-defensins are structurally
and functionally distinct. Human
-defensin HNP-2 is a
noncovalent dimer, but rabbit NP-1 is monomeric (11, 12). Dimeric HNP-2 forms stable multimeric pores after insertion into model membranes (13), and a pore model in which six HNP-2 dimers intercalate in the
bilayer to form the 20 Å pore annulus has been proposed based on
fluorescence leakage studies with large unilamellar vesicles (LUV)
(14). In contrast, NP-1 does not induce stable multimeric pores, but
evidence shows that this peptide permeabilizes LUV by creating large,
short-lived defects in the model membrane (15). Thus, myeloid
-defensins from human and rabbit neutrophils achieve bacterial cell
killing by highly distinctive membrane disruptive mechanisms.
Preliminary findings show that Crp4 induces graded leakage of
fluorophores from LUV, but the mechanisms by which individual
Paneth cell
-defensins achieve microbial cell killing are
unexplored. As HNP and NP comparisons reveal, the means by which Paneth
cell
-defensins exert bactericidal effects cannot be extrapolated
from existing literature or from a single model peptide.
-defensin (4, 5, 22) and because the molecular and
cellular details of Crp4 activation from inactive pro-Crp4 by MMP-7
proteolysis are known (10).3
Furthermore, Crp4 has a unique structure among the known
-defensins in that its polypeptide backbone contains a
three-residue deletion in the loop formed by the
Cys3-Cys5 disulfide bond (4, 5, 22).
-helical peptide (23). Also, the lack of pro-Crp4 microbicidal
activity and the inhibitory action of the precursor proregion on Crp4
bactericidal activity are consistent with the inability of pro-Crp4 and
proregion-Crp4 mixtures to interact with these model membranes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
-D-1-thiogalactopyranoside, and bacterial
cells were harvested by centrifugation and stored at
20 °C after
growth at 37 °C for 6 h. The cells were lysed by resuspending
bacterial cell pellets in 6 M guanidine HCl in 100 mM Tris-HCl, pH 8.1, followed by sonication at 70% power,
50% duty cycle for 2 min using a Branson Sonifier 450. The lysates
were clarified by centrifugation in a Sorvall SA-600 rotor at
30,000 × g for 30 min at 4 °C prior to protein purification.
mutant of Salmonella typhimurium.
Bacteria growing exponentially at 37 °C in trypticase soy broth were
deposited by centrifugation at 1700 × g for 10 min,
washed in 10 mM PIPES (pH 7.4) and resuspended in 10 mM PIPES (pH 7.4) that was supplemented with 0.01 vol of prepared trypticase soy broth. The peptide samples were lyophilized and
dissolved in 10 mM PIPES (pH 7.4) at 1 mg/ml. The
microorganisms were incubated at 37 °C with peptides in a total
volume of 50 µl at a concentration of ~1 × 106/ml
for 1 h in a shaking incubator. Following microbial exposure to
peptides, 20-µl samples of peptide-exposed bacteria were diluted 1:200 with 10 mM PIPES (pH 7.4), and 50 µl of the diluted
samples were plated on trypticase soy agar plates using an Autoplate
4000 (Spiral Biotech Inc., Bethesda, MD). The surviving microorganisms were counted as colony-forming units/ml after incubation at 37 °C
for 12-18 h.
where PB = Ablue/(Ablue + Ared), and A is the absorbance at 640 nm, the "blue" component of the spectrum, or at 500 nm, the "red" component. Note that blue and red refer to the visual
appearance of the material, not actual absorbances.
PB0 is the blue/red ratio of the control sample
before induction of a color change, and PBI is
the value obtained for the vesicle solution after the colorimetric transition occurred.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Bactericidal activities of Crp4, pro-Crp4,
and G1W-Crp4. A, the primary structures of recombinant
peptides prepared and investigated in these studies. MMP-7 cleavage
sites in the pro-Crp4 proregion, disclosed by protein sequencing of
MMP-7 pro-Crp4 digests, are noted by downward arrows.
Numerals below the pro-Crp4 sequence refer to residue
positions at the beginning of N-terminal sequences detected in the
digests, numbering with the initiating Met residue in prepro-Crp4 as
residue position 1. The Crp4 and G1W-Crp4 peptide sequences are shown
aligned with the Crp4 portion of pro-Crp4. In B, 1-µg
samples of purified recombinant Crp4 (lane 1), G1W-Crp4
(lane 2), and pro-Crp4 (lane 3) were resolved by
acid-urea PAGE and stained with Coomassie Blue (38). The
arrows at left indicate, in descending order, the
electrophoretic positions of pro-Crp4, Crp4, and G1W-Crp4. In
C, exponentially growing bacterial cells were exposed to
peptides at the indicated concentrations for 1 h, and the
surviving bacteria were quantitated ("Experimental Procedures"). In
both panels, the symbols denote surviving bacteria following exposure
to pro-Crp4 (triangles), Crp4 (filled circles),
or G1W-Crp4 (open circles). The upper panel shows
killing curves against S. typhimurium PhoP ,
and the lower panel shows killing of E. coli
DH5
cells.
mutant of S. typhimurium and
the DH5
strain of E. coli. As expected, Crp4 and G1W-Crp4
showed extensive bactericidal activity in the low micromolar range,
equal to 5-10 µg/ml of peptide (Fig. 1C). The
bactericidal activities of both peptides were the same against S. typhimurium PhoP
(Fig. 1C, upper
panel). Against E. coli, the G1W-Crp4 variant was
slightly less active, requiring 3-5-fold greater peptide
concentrations to achieve killing equivalent to that of Crp4 (Fig.
1C, lower panel). In contrast to the bactericidal
activities of these mature Crp4 peptides (5, 20, 22), pro-Crp4 lacked
microbicidal activity (Fig. 1C). This finding is consistent
with published (9, 10, 21) and recent3 studies showing that
Crp precursors lack bactericidal activity until activated by cleavage
with MMP-7. Because pro-Crp4 lacks bacterial cell killing activity, it
provided a useful reagent for testing whether interactions with model
membranes correlate with bactericidal activity. The bactericidal
activity of G1W-Crp4 similarly validates the use of this peptide as a
biologically relevant fluorescent probe (see below).
-defensin consisting of three
-strands and no
-helical
content. In contrast, melittin, is a potently bactericidal, membrane
disruptive peptide that assumes an amphipathic
-helical structure in
hydrophobic environments (23, 25). Interactions between melittin and
model membranes have been studied extensively (26-30). Prior to
comparisons of their membrane interaction dynamics, the bactericidal
activities of Crp4 and melittin also were assayed against E. coli (Fig. 2). As anticipated, the
bactericidal activity of melittin exceeds that of Crp4 against E. coli ML35, although both peptides are active in the low micromolar
range (Fig. 2). The different antibacterial activities of these two
peptides provide the biological basis for direct comparisons of the
mechanisms by which these two molecules interact with model
membranes.
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Fig. 2.
Relative bactericidal activities of Crp4 and
melittin against E. coli. Exponentially growing E. coli ML35 cells were exposed to peptides at the concentrations
shown in 10 mM PIPES (pH 7.4), 1% Trypticase Soy Broth for
1 h at 37 °C ("Experimental Procedures"). Following
exposure, the bacteria were plated onto Trypticase Soy Agar plates,
incubated for 16 h at 37 °C, and the surviving bacteria were
quantitated as colony-forming units/ml (cfu/ml,
"Experimental Procedures"). Colony-forming units/ml values below
1 × 103 indicate that no CFU were detected on the
plate. Filled circles, Crp4; empty circles,
melittin.
-defensin interactions with LUV composed of
bacterial membrane phospholipids have correlated microbicidal activity
with affinity for the membrane lipids (31). Accordingly, the binding
and interactions of Crp4 with lipid bilayers were characterized using
colorimetric lipid/PDA membrane assays and compared with the behavior
of melittin. To first validate the colorimetric lipid/PDA assay for
analysis of Crp4-membrane interactions, colorimetric titration curves
were performed to test whether the blue-red transitions were dependent
on peptide concentration and correlated with the phospholipid content
in DMPC/PDA mixed vesicles (Fig. 3). The
calculated %CR curves ("Experimental Procedures") within the
vesicle solutions determined at different peptide concentrations showed
that the extent of colorimetric transitions depend upon the DMPC/PDA
ratio within the vesicles. In the presence of 1 µM Crp4,
the %CR approached 80% when the DMPC/PDA molar ratio was 2:3, but
lower %CR values of ~40 and ~20% were observed when the DMPC/PDA
molar ratio of the mixed vesicles was reduced to 1:4 and 1:9,
respectively (Fig. 3). These findings demonstrate that the observed
chromatic transitions result from specific interactions between Crp4
and the phospholipid molecules incorporated into the PDA matrices and
not from nonspecific associations of the peptide with the PDA polymeric
matrix itself (17).
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Fig. 3.
Correlation between lipid content and
concentration in Crp4-induced blue-red transitions. %CR values
were calculated from measurements of chromatic shifts induced by Crp4
in DMPC/PDA vesicles ("Experimental Procedures"). The individual
curves show %CR values for mixed vesicles prepared from the following
proportions of DMPC to PDA. Circles, 10% DMPC/90% PDA;
triangles, 20% DMPC/80% PDA; squares, 40%
DMPC/60% PDA.
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Fig. 4.
Relative binding of Crp4 and melittin to
lipid/PDA vesicles. The slopes of individual curves are indicative
of the relative affinities of Crp4 (triangles) and melittin
(squares) for the lipid/PDA vesicles. Plateaus correspond to
maximum concentration of bound peptides. A, vesicles
composed of DMPC/PDA (2:3 mole ratio); B, vesicle composed
of DMPG/DMPC/PDA (1:1:3 mole ratio).
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Fig. 5.
%CR transitions induced by Crp4 and melittin
in vesicles of different lipid composition. Vesicle solutions
containing DMPC/PDA (2:3 mole ratio, A), DMPG/DMPC/PDA
(1:1:3 mole ratio, B), or total lipid extracts from
E. coli (C) were exposed to Crp4
(triangles) or melittin (squares) as described
under "Experimental Procedures." The relative increase in %CR is
related to depth of peptide insertion into the lipid layer, showing
that melittin inserts more deeply into the lipid bilayer.
1 µM (Fig. 5). The
Crp4 %CR values are 20-50% higher than those induced by melittin, an
indication that Crp4 is located predominantly at the lipid/water interface, causing enhanced perturbation in the head group region of
the lipid/polymer assembly (16). Melittin, on the other hand, inserted
more deeply into the hydrophobic core of the lipid bilayer and
consequently induced lower %CR values (Fig. 5). Previous colorimetric analyses have shown that melittin penetrates substantially into the
hydrophobic acyl chain region in lipid/PDA vesicle assemblies (16). For
example, a comparative study between melittin and a diastereomer analog
that does not adopt helical structure and attach at the lipid/water
interface has clearly shown a higher colorimetric response for the
latter melittin analog relative to the native peptide (16).
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Fig. 6.
Differential sensitivity of Crp4 and melittin
interactions to vesicle lipid composition. The degree of blue-red
transitions (%CR) induced by Crp4 (A) and melittin
(B) were measured in mixed vesicles of differing lipid
compositions. open circles, LPS/DMPC/PDA (0.2:2:3 mole
ratio); squares, DMPC/PDA (2:3); triangles,
DMPG/DMPC/PDA (1:1:3); filled circles, E. coli
lipids/PDA (2:3 weight ratio). Crp4-lipid interactions are sensitive to
the composition of the bilayer, but the %CR values for melittin are
affected less by the vesicle lipid contents examined.
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Fig. 7.
Trp fluorescence emission shifts of peptides
in different vesicles. Trp fluorescence emission shifts induced by
G1W-Crp4 (A) and melittin (B) were measured in
DMPG/DMPC/PDA vesicles (1:1:3 mole ratio, triangles) and
DMPC/PDA vesicles (2:3 mole ratio, squares). The
fluorescence shifts correlate with greater depth of melittin
incorporation within the hydrophobic environment of the lipid acyl
chains.
-defensin peptides
(10).3 To determine whether the lack of pro-Crp4
bactericidal activity correlates with an inability of the precursor to
perturb membranes, interactions between pro-Crp4 and Crp4 with
lipid/PDA vesicles were compared using DMPG/DMPC/PDA vesicles and found
to be markedly different (Fig.
8A). In contrast to the
~50%CR induced by mature Crp4, pro-Crp4 induced a value of
<5%CR, evidence that interactions between pro-Crp4 and lipid/PDA
assemblies were negligible. These findings are consistent with
preliminary studies of Crp4 and pro-Crp4-induced graded fluorophore
leakage from palmitoyl-oleoyl-phosphatidyl glycerol
LUV.4
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Fig. 8.
Attenuation of Crp4-membrane interactions by
proregion. In A, %CR measurements of interactions
between 1.3 µM Crp4 and pro-Crp4 and vesicles composed of
DMPC/PDA (2:3 mole ratio) were taken ("Experimental Procedures").
In B, the %CR values represent the interactions between
Crp4 and Crp4 prosegment mixtures at several Crp4 to proregion molar
ratios. In C, Trp emission shifts were measured for G1W-Crp4
and for G1W-Crp4 prosegment mixtures at the indicated molar ratios.
Colorimetric and Trp fluorescence measurements demonstrate that
pro-Crp4 associates poorly with membranes and that the Crp prosegment
prevents of membrane interactions of when added to mature Crp4 in
trans.
-defensins from human
neutrophils (37) and mouse Paneth cells (10). Accordingly, the effects
of added prosegment on Crp4 membrane interactions were evaluated. The
addition of increasing quantities of full-length Crp1 prosegment to
Crp4 prior to mixing with the lipid/PDA vesicles ("Experimental
Procedures") inhibited the Crp4-induced %CR to low levels similar to
those induced by pro-Crp4 (Fig. 8B). Prosegment alone
induced no %CR (data not shown). The extent to which the colorimetric
response was blocked related directly to the molar ratio of prosegment to Crp4 in the mixtures. At the highest Crp4:prosegment ratio tested
(11:1), the %CR recorded was nearly 40%, similar to the 48% CR value
obtained with intact Crp4 alone (Fig. 8B). At increasingly higher prosegment to Crp4 molar ratios, %CR diminished progressively to a nadir of 5% at a Crp4/prosegment ratio of 0.6 (Fig.
8B).
-sheet CD
spectrum (Fig. 9, solid line). On the other hand, the CD
spectra for both pro-Crp4 (Fig. 9, short dashes) and
Crp4-prosegment mixtures at 0.6:1 molar ratios differ and are
indicative of random coil peptide conformations (Fig. 9,
short and long dashes, respectively). These CD
signatures show that the
-sheet trace is attenuated in pro-Crp4 and
in the noncovalent Crp4-prosegment complex. This was particularly
evident when prosegment was added in trans and when the CD
trace represents the difference spectrum obtained after the prosegment
CD trace was subtracted (Fig. 9, long dashed tracing),
depicting primarily the Crp4 signature. These data suggest that one
possible mechanism for prosegment inhibition of Crp bactericidal
activity involves destabilization or attenuation of the
-sheet
topology, perhaps via electrostatic interactions that disrupt the
ability of the Crp4 molecule to perturb target cell membranes.
View larger version (14K):
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Fig. 9.
Destabilization of Crp4 structure by the
proregion. CD data spectra indicate that the -sheet structure
of native Crp4 is attenuated in pro-Crp4 and after addition of
prosegment to Crp4 in trans. CD traces were recorded in
aqueous solutions containing DMPC/PDA vesicles (2:3 mole ratio,
"Experimental Procedures"). Solid line, mature Crp4;
long dashes, pro-Crp4; short dashes, Crp4
combined with equimolar ratio of prosegment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-defensins
to permeabilize LUV being dependent on vesicle lipid composition (31)
and with studies of Crp4-induced fluorophore leakage from LUV prepared
from palmitoyl-oleoyl-phosphatidyl glycerol or LUV made from 4:1
palmitoyl-oleoyl-phosphatidyl glycerol:palmitoyl-oleoyl-phosphatidyl choline.4 For example, spectroscopic data (Fig. 7) showed
that the depth of Crp4 insertion is favored by the presence of
negatively charged phospholipids in the lipid bilayer and that
zwitterionic phospholipids and LPS promote greater interfacial binding.
Colorimetric and Trp fluorescence results are consistent in indicating
deep penetration of Crp4 into PDA vesicles consisting of E. coli total cell lipids (Figs. 5 and 6). This finding is somewhat
unexpected because LPS is an integral component of Gram-negative
bacterial cell envelope, although it seems unlikely that the complex
orientation and organization of the membrane-associated LPS is retained
in the E. coli lipids/PDA vesicle environment.
-sheet structure of Crp4 is attenuated in both pro-Crp4 or when
prosegment was added to Crp4 in trans (Fig. 9). Although the
individual residues that associate to cause the inhibition are not
known, it seems likely that the 13 anionic side chains of the proregion
interact with and neutralize the seven cationic side chains on the Crp4
peptide surface, thus inhibiting electrostatic interactions at the
target cell membrane. Collectively, these findings are consistent with
a model in which MMP-7 cleavage of the pro-Crp4 proregion allows Crp4
to assume a tertiary conformation that permits the peptide to associate
with and disrupt target cell membranes, thus activating bactericidal
peptide activity.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael E. Selsted for useful discussions, Dr. Agnes Henschen-Edman (UCI Biomedical Protein and Mass Spectrometry Resource Facility) for peptide sequencing and analysis of recombinant peptides, and Khoa Nguyen, Vungie Hoang, and Victoria Rojo for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by funds from the Israel-United States Binational Science Foundation (to R. J. and A. J. O.) and by National Institutes of Health Grants DK10184 (to D. P. S.) and DK44632 (to A. J. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to these studies.
¶ Present address: Biocides R&D, The Dow Chemical Company, Buffalo Grove, IL 60089.
Member of the Ilse Katz Center for Nanoscience and Technology.
To whom correspondence should be addressed: Dept. of Chemistry, Ben-Gurion University of the Negev, Beersheva, Israel 84105. Tel.: 972-8-6461747; Fax: 972-8-6472943; E-mail:
razj@bgumail.bgu.ac.il.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M212115200
2 M. Katz, H. Tsubery, M. Fridkin, S. Kolusheva, and R. Jelinek, submitted.
3 Shirafuji, Y., Tanabe, H., Satchell, D. P., Henschen-Edman, A., Wilson, C. L., and Ouellette, A. J. (2003) J. Biol. Chem. 278, 7910-7919.
4 J. Cummings, D. P. Satchell, T. K. Vanderlick, and A. J. Ouellette, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: Crp, cryptdin; LUV, large unilamellar vesicle(s); PDA, polydiacetylene; DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol; LPS, lipopolysaccharide; pro-Crp, procryptdin-4; RP-HPLC, reverse phase high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization mode time-of-flight; PIPES, 1,4-piperazinediethanesulfonic acid; %CR, percentage colorimetric response.
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