Alanine Scanning Studies of the Antimicrobial Peptide Aurein 1.2

Article abstract of DOI:10.1007/s12602-018-9501-0

Antimicrobial peptides (AMPs) are compounds widely distributed in nature that display activity against a broad spectrum of pathogens. Amphibian skin, as an organ rich in pharmacologically active peptides, appears to be an interesting source of novel AMPs. Aurein 1.2 (GLFDIIKKIAESF-NH₂) is a short 13-residue antimicrobial peptide primarily isolated from the skin secretions of Australian bell frogs. In this study, the alanine scan of aurein 1.2 was performed to investigate the effect of each amino acid residue on its biological and physico-chemical properties. The biological studies included determination of minimum inhibitory concentration, activity against biofilm, and inhibitory effect on its formation. Moreover, the hemolytic activity as well as serum stability was determined. The hydrophobicity of peptides and their self-assembly were investigated using reversed-phase chromatography. In addition, their helicity was calculated from circular dichroism spectra. The results not only provided information on structure-activity relationship of aurein 1.2 but also gave insights into design of novel analogs of AMPs in the future.

Full text of DOI:10.1007/s12602-018-9501-0

Probiotics and Antimicrobial Proteins
https://doi.org/10.1007/s12602-018-9501-0
Alanine Scanning Studies of the Antimicrobial Peptide Aurein 1.2
Dorian Migoń1,2 & Maciej Jaśkiewicz1 & Damian Neubauer1 & Marta Bauer1 & Emilia Sikorska3 & Elżbieta Kamysz3
&
Wojciech Kamysz1
#
The Author(s) 2018
Abstract
Antimicrobial peptides (AMPs) are compounds widely distributed in nature that display activity against a broad spectrum of
pathogens. Amphibian skin, as an organ rich in pharmacologically active peptides, appears to be an interesting source of novel
AMPs. Aurein 1.2 (GLFDIIKKIAESF-NH2) is a short 13-residue antimicrobial peptide primarily isolated from the skin secretions
of Australian bell frogs. In this study, the alanine scan of aurein 1.2 was performed to investigate the effect of each amino acid
residue on its biological and physico-chemical properties. The biological studies included determination of minimum inhibitory
concentration, activity against biofilm, and inhibitory effect on its formation. Moreover, the hemolytic activity as well as serum
stability was determined. The hydrophobicity of peptides and their self-assembly were investigated using reversed-phase chro-
matography. In addition, their helicity was calculated from circular dichroism spectra. The results not only provided information on
structure-activity relationship of aurein 1.2 but also gave insights into design of novel analogs of AMPs in the future.
.
.
.
.
.
Keywords Aurein 1.2 Antimicrobial peptides Structure-activity relationship Antimicrobial agents Peptide drugs
Antibiotics
Introduction
needed. Antimicrobial peptides (AMPs) as a class of com-
pounds meeting those requirements offer an interesting alter-
native to conventional antibiotics. AMPs are endogenic mol-
ecules widely distributed in nature, mainly as a part of innate
immune system. Generally, they are 11–50 amino acid residue
long, amphipathic molecules with net positive charge at neu-
tral pH [3]. The main mechanism of their antimicrobial activ-
ity is based on a non-receptor-mediated microbial membrane
disruption. Moreover, AMPs lead to microbial cell death via
other mechanisms such as inhibition of protein/DNA or cell
wall synthesis or induction of apoptosis/necrosis. Owing to
those properties, AMPs display antimicrobial activity against
a broad spectrum of pathogens such as Gram-positive and
Gram-negative bacteria, fungi, protozoa, and viruses.
Because of the membrane-associated activity, they could also
exhibit a low potency to trigger antimicrobial resistance [4].
Amphibian skin, as an organ rich in pharmacologically active
peptides, appears to be an interesting source of novel AMPs.
Physiologically, it is responsible for regulation of biological
fluids equilibrium, respiration, and transportation of essential
ions. To assure efficient and unlimited course of these process-
es, amphibian skin is adequately thin and humid. This in turn
increases the risk of infection. Because of that, amphibians
had evolutionarily developed a series of defense mechanisms,
with AMP biosynthesis being one of them [5, 6]. Until now,
about 1000 amphibian AMPs have been isolated and
An increasing resistance to antibiotics among microorganisms
looms a serious threat to human society. According to the
Review on Antimicrobial Resistance, at least 700,000 annual
deaths are caused by drug-resistant infections. Furthermore,
this number was estimated to soar up to 10 million in 2050, if
only the current antibiotic politics remain unchanged [1].
There are several reasons of this situation and due, among
others, to inappropriate antibiotic prescribing, extensive agri-
cultural use, and limited access to new antibiotics [2]. In this
situation, the development of antimicrobial agents with im-
proved modes of action and low-resistance potential is
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s12602-018-9501-0) contains supplementary
material, which is available to authorized users.
* Dorian Migoń
dorianmig@gumed.edu.pl
1
Department of Inorganic Chemistry, Faculty of Pharmacy, Medical
University of Gdańsk, Al. Gen. J. Hallera 107,
80-416 Gdańsk, Poland
2
Polpharma Biologics, Gdańsk, Poland
3
Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland

Probiotics & Antimicro. Prot.
examined [7]. However, because of species multiplicity, their
total number could reach even 100,000 individual compounds
[8]. Unfortunately, AMPs are usually chemically and biolog-
ically unstable, and relatively toxic towards human cells.
However, chemical synthesis of analogs based on those
AMPs can lead to more stable and active candidates for anti-
microbials [9]. One of the tools used for a better understanding
of peptide activity is alanine scanning. This approach allows
to determine the contribution of each amino acid residue to
specific peptide features what helps to rationalize further mod-
ifications. Aurein 1.2 (GLFDIIKKIAESF-NH2) is a short 13-
residue antimicrobial peptide with molecular mass of approx-
imately 1480 Da and positive (+ 1) net charge at physiological
pH. It was initially isolated from Australian bell frogs,
Ranoidea aurea (formerly Litoria aurea) and Ranoidea
raniformis (formerly L. raniformis) skin secretions [10].
Antimicrobial activity of aurein 1.2 ranges between 1 and
16 μg/mL in terms of minimum inhibitory concentrations
(MICs) against both reference and clinical strains of Gram-
positive bacteria such as Staphylococcus aureus,
Enterococcus faecalis, or Streptococcus pyogenes (including
methicillin-resistant S. aureus and vancomycin-resistant
E. faecalis). Moreover, a synergistic effect between aurein
1.2 and minocycline/clarithromycin against those bacteria
has also been reported [11]. On the other hand, in vitro studies
demonstrated aurein 1.2 to exhibit a low antimicrobial activity
against Escherichia coli and Pseudomonas aeruginosa (MIC
values for both strains are 256 μg/mL) [12] and moderate
activity against Candida albicans strains (MIC value of
32 μg/mL) [13]. Additionally, anticancer activity of aurein
1.2 has been demonstrated through the NCI-60 human tumor
cell lines [14]. It exerts cytotoxic activity against several hu-
man neoplastic cell lines of leukemia and melanoma as well as
the lung, colon, central nervous system ovarian, prostate, and
breast cancer cell lines [10]. Aurein 1.2 does not adopt any
secondary structure in aqueous media. After incorporation in-
to the lipid membrane, it adopts an α-helical geometry.
Fernandez. et al. demonstrated that aurein 1.2 exerts disruptive
activity via carpet-like mechanism against both neutral and
anionic model membranes, with a greater affinity towards
the latter [15]. Furthermore, significant alternation of lipid
components between the negatively charged model mem-
brane leaflets due to aurein 1.2 binding has also been demon-
strated [16]. Laadhari et al. in their in vivo studies suggest that
antimicrobial activity of aurein 1.2 is not only an effect of
membrane destabilization and/or disruption, but also interac-
tions between the peptide and bacterial teichoic acid and
lipoteichoic acid should be considered [17].
and the tendency to self-association. The results not only pro-
vided the information on the structure-activity relationship of
aurein 1.2 but also gave insights into design of novel analogs
of AMPs in the future.
Materials and Methods
Peptide Synthesis
Aurein 1.2 and its analogs were synthesized manually accord-
ing to the standard Nα-Fmoc protocol on a Rink Amide resin
(0.7 mmol/g substitution). Peptide chains were elongated
using repeated cycles of deprotection and acylation.
Deprotection of Fmoc groups was conducted using a 20%
piperidine solution in DMF (1 × 20 min). Coupling reactions
were carried out using 3 eq. of protected amino acids (Fmoc-
AA), Oxyma Pure, and DIC (1:1:1) dissolved in DCM/DMF.
Peptide cleavage and side-chain deprotection were accom-
plished in 1 h through a one-step procedure using a mixture
of TFA/TIS/H2O (95:2.5:2.5). The crude peptides were pre-
cipitated with cold anhydrous diethyl ether and lyophilized.
Subsequently, the peptides were purified by reversed-phase
high-performance liquid chromatography (RP-HPLC) with
LP-chrom software. Purifications were carried out on a
Phenomenex Gemini-NX C18 column (21.20 × 100 mm,
5-μm particle size, 110-Å pore size). UV detection at
214 nm was used, and the crude peptides were eluted with a
linear 10–60% acetonitrile gradient in deionized water over
60 min at room temperature. The mobile phase flow rate was
10.0 mL/min. Both eluents contained 0.1% (v/v) of TFA.
Fractions were analyzed on a Waters XBridge Shield RP-18
column (4.6 × 150 mm, 3.5-μm particle size, 130-Å pore size)
with UV detection at 214 nm. Pure fractions (> 95%, by
HPLC analysis) were collected and lyophilized. The molecu-
lar weights were confirmed by ESI-MS.
Antimicrobial Activity
The minimum inhibitory concentrations (MICs) of test com-
pounds were assessed by broth microdilution method on 96-
well plates, according to the guidelines of Clinical and
Laboratory Standards Institute (CLSI) [18, 19]. For this pur-
pose, reference strains of bacteria: S. aureus ATCC 25923,
E. faecalis PCM 2673, E. coli ATCC 25922, and
P. aeruginosa ATCC 9027; and fungus: C. albicans ATCC
10231 were used (The Polish Collection of Microorganism,
Polish Academy of Sciences, Wrocław, Poland). Bacteria at
initial inoculums of 0.5 × 105 CFU/mL in Mueller Hinton
Broth (MHB), and fungi at initial inoculums of 2 ×
103 CFU/mL in RPMI-1640, were exposed to the compounds
over the concentration range of 1–512 μg/mL. The polysty-
rene plates were incubated at 37 °C for 18 h with bacteria and
In this study, alanine scanning of aurein 1.2 was performed
to recognize the effect of each amino acid residue on its bio-
logical and physico-chemical properties. For this purpose,
multiple parameters were measured, including the following:
hydrophobicity, helicity, biological activity, serum stability,

Probiotics & Antimicro. Prot.
for 24 h with C. albicans. The MIC was taken as the lowest
concentration at which a noticeable growth of microorganisms
was inhibited. Minimum biofilm eradication concentrations
(MBECs) were determined using a previously described pro-
tocol [20]. For this purpose, 96-well polystyrene flat-bottom
plates and a resazurin (7-hydroxy-3H-phenoxazin-3-one 10-
oxide) were used. In this assay, a specific feature of resazurin
is utilized, which upon the contact with living cells is metab-
olized and reduced from the basic blue form to pink resorufin.
Briefly, 24-h cultures of microorganisms were diluted 1,000-
fold and 100 μL of that suspension was added into the test
plates. After 24 h of incubation at 37 °C, the wells of the plates
were rinsed three times with phosphate buffer saline (PBS) to
remove non-adherent cells. Subsequently, 100 μL of test com-
pounds in concentration range (diluted in appropriate media)
were added to each well. After 24 h of incubation at 37 °C,
20 μL of the resazurin (4 mg/mL) was added. The MBEC was
read after 1 h. Additionally, anti-adhesive properties of the
compounds were also analyzed. The assay was conducted
on 96-well polystyrene flat-bottom plates. The preparation
of inoculums was followed by a 500-fold dilution of 24-h
cultures of microorganisms. Briefly, to evaluate the effect of
the peptides in preventing biofilm formation, 50 μL of com-
pounds in concentration range, diluted in appropriate medium,
were prepared. Subsequently, the 50 μL of the bacterial/fungal
inoculums were added to reach the same microbial density as
that in MBEC assay. After 24 h of incubation at 37 °C, the
wells were rinsed three times with PBS and the fresh media
with resazurin (0.6 mg/mL) were added. Then, after 1 h of
incubation, the minimum biofilm inhibitory concentrations
(MBIC) were read. MBECs as well as MBICs were deter-
mined as the lowest concentration at which the reduction of
resazurin was lower or equal (10% 0.5%) as compared to
positive (100%) and negative (0%) controls. All experiments
were performed in triplicate.
the release of hemoglobin was measured at 540 nm
(Multiskan™ GO Microplate Spectrophotometer, Thermo
Scientific). All experiments were conducted in triplicate.
Determination of Peptide Hydrophobicity Parameter
The hydrophobicity parameter was expressed as RP-HPLC
mobile phase acetonitrile content (%ACN) at a retention time
of a particular peptide. All peptides were analyzed using
Waters Alliance e2695 system with a Waters 2998 PDA
Detector (software–Empower®3). All HPLC analyses were
conducted in triplicate on a Waters XBridge™ Shield RP-18
column (4.6 × 150 mm, 3.5-μm particle size, 130-Å pore size)
using a linear 30–60% ACN (B) gradient in deionized water
(A) over 30 min (1% ACN/min) at 25.0 0.1 °C, at a flow rate
of 0.5 mL/min and UV detection at 214 nm (A–0.1% aqueous
TFA and B–0.1% TFA in ACN).
Determination of Peptide Self-Association
Peptide molecules exist in solution as monomeric and/or self-
associated species. In most cases, peptide self-association oc-
curs through hydrophobic interactions between their non-
polar faces. Interestingly, in order to interact with hydrophobic
stationary phase, peptide molecule needs to be in its mono-
meric form (i.e., needs to have its non-polar face unoccupied).
Because of this, self-associated species disruption, being a
result of temperature increase, result in longer retention time
of peptide. On the other hand, increasing temperature causes
also a decrease in mobile phase viscosity and increase in mass
transfer between the mobile and stationary phase. These, in
turn, contribute to accelerated retention and stand in contrast
to the effect caused by disruption of self-associates. Peptide
tendency to self-association may be characterized by a maxi-
mum (Tmax) on a normalized plot of the change in retention
time at different temperatures. At Tmax, the impact of these
different effects on retention time is equal [22]. Self-
association of the peptides was determined using RP-HPLC
temperature profiling analyses. All peptides were analyzed
using Waters Alliance e2695 system with a Waters 2998
PDA Detector (software–Empower®3). The temperature
ranged from 5 to 60 °C at 5 °C increments ( 0.1 °C).
Analyses were performed in triplicate at each temperature
using conditions described in detail in the preceding section.
Peptide retention times were normalized by subtraction with
the retention time value obtained at 5 °C and plotted over
adequate temperature values. Peptide self-association tenden-
Hemolysis Assay
The hemolytic activity of the peptides was determined using
fresh human red blood cells (RBCs) collected from a healthy
human donor according to the procedure described in the liter-
ature [21]. Fresh RBCs with EDTA as anticoagulant were
rinsed three times with a PBS by centrifugation at 800×g for
10 min and resuspension in PBS. Serial dilution of the peptides
in PBS (1–256 μg/mL) on 96-well polystyrene plates was pre-
pared and the stock solution of RBCs was added up to a final
volume of 100 μL with a 4% concentration of erythrocytes (v/
v). The control wells for 0 and 100% hemolysis contained of
RBCs suspended in PBS and 1% of Triton-X 100, respectively.
Subsequently, the plates were incubated for 1 h at 37 °C and
then centrifuged at 800×g for 10 min at 4 °C (Sorvall ST 16R
Centrifuge, Thermo Scientific). After centrifugation, the super-
natant was carefully resuspended to new microtiter plates and
cy was expressed as Tmax
.
Serum Stability
To assess serum stability, the peptides (0.5 mg) were dissolved
in 4 mL of 25% (v/v) fetal bovine serum (FBS) in PBS and

Probiotics & Antimicro. Prot.
incubated at 37 °C. The aliquots of 100 μL of the test samples
were transferred to 96-well polystyrene plates in triplicate for
each time point. Samples were quenched at zero time (t0) and
after 6 h of incubation at 37 °C by addition of 100 μL of ice-
cold acetonitrile and stored at 4 °C for 10 min. Afterwards, the
samples were centrifuged at 13,000×g for 20 min at 4 °C and
diluted 1:1 with HPLC-grade water. All samples were ana-
lyzed using the Waters Alliance e2695 system with Waters
Acquity QDa Detector (software–Empower®3). Analyses
were conducted on an Agilent Zorbax SB-C18 RP-18 column
(4.6 × 50 mm, 1.8-μm particle size, 80-Å pore size) using a
linear 30–60% acetonitrile (B) gradient in deionized water (A)
over 6 min at 25.0 0.1 °C, at a flow rate of 0.5 mL/min and
MS working in selected ion recording (SIR) mode (A—0.1%
aqueous formic acid (FA) and B—0.1% FA in acetonitrile).
The percentage of peptide remaining intact was calculated
from peak areas of samples at t0 and after 6 h of incubation
in serum (the % of remaining peptide = peak area after 6 h/
peak area at t0). The mass-to-charge ratios selected for SIR
mode were based on values obtained from full spectrum anal-
ysis of each peptide and are presented in Table 1.
it exhibited only the order of magnitude higher activity, while
with C. albicans the opposite relation was found. In this case,
D4A exhibited the order of magnitude lower activity. Another
compound was E11A, with the MIC values from 1- to 2-fold
dilutions lower against all bacterial strains and the same for
C. albicans. For S12A, the higher activity was found only for
S. aureus and the same for other strains with an exception for
P. aeruginosa. For this strain, the growth was not inhibited at a
whole concentration range. The remaining compounds were
either ineffective or their MIC values were higher than or
equal to 128 μg/mL with an exception for K8A and
C. albicans. For this compound, the MIC value was a magni-
tude higher than for aurein 1.2. The MBEC values indicate
that only D4A eradicated biofilm of all the test strains at con-
centrations used in the study (Table 3). The highest activity
was found against S. aureus with MBEC value of 16 μg/mL,
which was 4-fold lower than that of aurein 1.2. Identical result
against S. aureus biofilm was found for E11A. Moreover, both
compounds eradicated biofilm formed by E. coli at 32 and
64 μg/mL and P. aeruginosa at 512 μg/mL, respectively. It
is noteworthy that analog S12A was as active as parent mol-
ecule against C. albicans biofilm, while other analogs exhib-
ited higher MBEC. Furthermore, D4A and S12A were the
only two compounds active against the E. faecalis biofilm
(128 μg/mL). Interestingly, all the test compounds exhibited
high anti-adhesive properties against C. albicans (Table 4);
but, the highest activity was found for aurein 1.2, D4A, and
S12A (2 μg/mL). D4A and E11A displayed high anti-
adhesive properties for all the test bacterial strains, and these
analogs were even more potent than aurein 1.2. Despite the
fact that S12A was more effective for S. aureus and E. coli, it
did not affect the biofilm formation of P. aeruginosa.
CD Measurements
The CD spectra of aurein 1.2 and its alanine scanning analogs
were recorded on a JASCO J-815 spectropolarimeter at 25 °C
over the 190–260-nm range under three different conditions,
i.e., surfactant-free phosphate buffer at pH 7.4, buffered
20 mM SDS (sodium dodecyl sulfate), and 20 mM DPC
(dodecylphosphocholine) micellar solutions. The peptide con-
centration was 0.15 mg/mL. Every spectrum was scanned three
times to amplify the signal-to-noise ratio. The spectra were
plotted against the mean residual molar ellipticity (MRME,
deg. × cm2 × dmol−1) versus wavelength (nm). Deconvolution
of the CD spectra was carried out using CDPro software with
CONTINILL algorithm and SMP56 database set [23].
Hemolysis Assay
Almost all the analogs were less hemolytically active against
human RBCs than aurein 1.2. Only K8A was the compound
with a right-shifted concentration curve, which means that at
almost all concentrations the hemolytic properties were higher
than that of the original compound. Among others, L2A, I6A,
I9A, and F13A did not cause the lysis of RBCs over the at
whole concentration range, while S12A was more hemolytic
between 64 and 16 μg/mL (Table 5).
Results
Peptide Synthesis
Sequences, average masses, net charges, and calculated and
measured mass-to-charge ratios of the synthesized peptides
are shown in Table 1.
Hydrophobicity Parameter of Peptides
Antimicrobial Assay
Hydrophobicity parameter of a specific peptide was expressed
as the percentage of acetonitrile in the mobile phase at the time
of peptide elution (Table 6). In general, substitution of the
amino acid residue containing a hydrophobic side chain (phe-
nylalanine, isoleucine, leucine) with alanine residue resulted
in peptides with depressed hydrophobicity parameter as com-
pared to that of aurein 1.2. With polar amino acid residues, the
Among the test compounds, only three aurein 1.2 analogs
exhibited satisfactory antimicrobial activity (Table 2). The
most effective was D4A with a 3-fold lower MIC values
against E. coli and S. aureus, and a 2-fold against
P. aeruginosa than the original compound. With E. faecalis,

Probiotics & Antimicro. Prot.
Table 1 Peptides synthesized in
this study
Peptide
Sequence
Average mass [Da]
Net charge
MS analysis
za
m/zb
m/zc
G1A
L2A
F3A
D4A
I5A
ALFDIIKKIAESF-NH2
GAFDIIKKIAESF-NH2
GLADIIKKIAESF-NH2
GLFAIIKKIAESF-NH2
GLFDAIKKIAESF-NH2
GLFDIAKKIAESF-NH2
1493.79
1437.68
1403.67
1435.75
1437.68
1437.68
+ 1
+ 1
+ 1
+ 2
+ 1
+ 1
3
2
3
2
3
2
3
2
3
2
3
2
2
2
3
2
3
2
3
2
3
2
3
2
498.93
747.90
480.23
719.84
468.89
702.84
479.58
718.88
480.23
719.84
480.23
719.84
712.34
712.34
480.23
719.84
474.91
711.86
488.92
732.88
468.89
702.84
494.25
740.88
498.98
747.90
480.31
719.64
468.95
702.63
479.58
718.79
480.36
719.69
480.34
719.76
712.31
712.15
480.36
719.84
474.92
711.57
488.90
732.72
468.91
702.69
494.32
740.74
I6A
K7A
K8A
I9A
GLFDIIAKIAESF-NH2
GLFDIIKAIAESF-NH2
GLFDIIKKAAESF-NH2
1422.67
1422.67
1437.68
0
0
+ 1
E11A
GLFDIIKKIAASF-NH2
GLFDIIKKIAEAF-NH2
GLFDIIKKIAESA-NH2
GLFDIIKKIAESF-NH2
1421.72
1463.76
1403.67
1479.76
+ 2
+ 1
+ 1
+ 1
S12A
F13A
aurein 1.2
a Ion charge
b Calculated mass-to-charge ratio;
c Measured mass-to-charge ratio, italicized values are mass-to-charge ratios selected for SIR LC-MS analysis
effect of the alanine residue substitution on peptide hydropho-
bicity was much more diverse. Substitution of either the lysine
or serine residues resulted in an increase of hydrophobicity
parameter. On the other hand, substitution of aspartic acid
residue only slightly increased peptide hydrophobicity, while
substitution of glutamic acid depressed it. Interestingly,
substituting glycine residue with alanine residue also resulted
in a peptide with a decreased hydrophobicity parameter. The
changes in hydrophobicity are fairly well correlated with ami-
no acid hydrophobicity scale derived from RP-HPLC analyses
(Table 6 and Supplementary Fig. 1). However, G1A and F3A,
unexpectedly, did not meet these predictions [24].
that of aurein 1.2. On the contrary, in every case of a peptide
with non-polar residue (phenylalanine, isoleucine, leucine)
substituted with the alanine residue, the self-association ten-
dency was suppressed. This phenomenon was distinct for I9A,
in which Tmax was half that compared to Tmax of aurein 1.2
(15 °C and 30 °C, respectively). Importantly, Tmax is not well
correlated with %ACN (simple linear regression, R2 = 0.631).
In general, if %ACN of the peptide used in this study was ≤
41.1%, then Tmax was ≤ 25 °C; and if %ACN was > 41.1%,
then Tmax was 30 °C (Supplementary Fig. 2).
Serum Stability of Peptides
Peptide Tendency to Self-Association
Serum stability of the peptides was expressed as percentage of
molecule which remained after 6-h incubation in FBS solution
at 37 °C (Table 7). Almost all peptides, with the exception of
S12A and I5A, were less stable than aurein 1.2. The most
stable molecule, S12A, retained 77% of initial concentration,
while the least stable molecules, L2A and I9A, were degraded
down to 6% of initial peptide concentration.
Generally, the tendency to self-association of the peptide was
influenced by altered amino acid residue hydrophobicity
(Table 6 and Supplementary Fig. 2). Substitution of any of
the polar amino acid residues with alanine residue did not
result in altered self-association tendency when compared to

Probiotics & Antimicro. Prot.
Table 2 The MIC (μg/mL)
values of the test compounds
against reference strains of
microorganisms. MIC values of
the analogs lower than those of
aurein 1.2 are italicized
MIC [μg/mL]
Peptide
S. aureus
E. faecalis
E. coli
P. aeruginosa
C. albicans
ATCC 25923
PCM 2673
ATCC 25922
ATCC 9027
ATCC 10231
G1A
L2A
512
128
512
256
32
128
256
128
8
512
128
256
128
64
> 512
512
512
F3A
512
D4A
I5A
16
64
> 512
> 512
> 512
> 512
> 512
32
128
256
512
64
256
512
> 512
128
512
16
512
256
256
> 512
64
I6A
> 512
> 512
> 512
> 512
64
K7A
K8A
I9A
128
32
512
32
E11A
S12A
F13A
urein 1.2
32
64
64
> 512
> 512
256
32
> 512
128
128
64
128
64
512
32
CD Results
adopt helical structure upon interaction with biological mem-
branes as expected for this class of antimicrobial compounds.
The content of helical structure predicted from the CD spectra
is displayed in the Table 8. As seen, the replacement of orig-
inally occurring amino acids with alanine residue reduced
helical structure content, except for two analogs, F3A and
K7A, in SDS solution, where a slight increase in helicity is
seen. Interestingly, Θ222/Θ208 ratio of all peptides in our
study was lesser or equal to 0.9. Therefore, regardless of used
surfactant, a coiled coil structure was adopted by none of
studied peptides.
CD spectra of aurein 1.2 and its alanine scanning analogs are
shown in Fig. 1. Due to poor solubility, the CD spectra of three
analogs (K7A, K8A, and S12A) could not be recorded in
surfactant-free phosphate buffer solution. The spectra of the
remaining peptides showed a minimum around 200 nm re-
vealing the state of random conformation. Addition of surfac-
tants, SDS or DPC, induced formation of a helical structure
with two characteristic minima at 208 and 222 nm in all the
CD spectra. This supports the hypothesis that the peptides
Table 3 The MBEC (μg/mL)
MBEC [μg/mL]
values of the test compounds
against reference strains of
microorganisms. MBEC values
of the analogs lower than those of
aurein 1.2 are italicized
Peptide
S. aureus
E. faecalis
E. coli
P. aeruginosa
C. albicans
ATCC 25923
PCM 2673
ATCC 25922
ATCC 9027
ATCC 10231
G1A
L2A
512
> 512
> 512
> 512
128
256
512
> 512
> 512
> 512
512
> 512
> 512
16
512
> 512
> 512
512
F3A
256
D4A
I5A
32
> 512
> 512
> 512
> 512
> 512
16
> 512
> 512
> 512
> 512
> 512
> 512
128
512
> 512
> 512
> 512
> 512
> 512
512
512
I6A
> 512
> 512
> 512
> 512
64
> 512
> 512
> 512
> 512
> 512
256
K7A
K8A
I9A
E11A
S12A
F13A
aurein 1.2
> 512
128
> 512
512
> 512
> 512
512
> 512
> 512
> 512
256
128
64

Probiotics & Antimicro. Prot.
Table 4 The MBIC (μg/mL)
values of the test compounds
against reference strains of
microorganisms. MBIC values of
the analogs lower than that of
aurein 1.2 are italicized
MBIC [μg/mL]
Peptide
S. aureus
E. faecalis
E. coli
P. aeruginosa
C. albicans
ATCC 25923
PCM 2673
ATCC 25922
ATCC 9027
ATCC 10231
G1A
L2A
512
256
128
512
256
8
256
4
8
4
2
4
4
4
4
16
4
2
8
2
> 512
>512
16
> 512
256
> 512
512
F3A
D4A
I5A
32
64
> 512
> 512
> 512
> 512
> 512
32
512
512
512
64
> 512
> 512
> 512
> 512
> 512
64
I6A
> 512
> 512
128
K7A
K8A
I9A
32
> 512
32
>512
16
E11A
S12A
F13A
aurein 1.2
32
128
32
>512
>512
128
>512
128
>512
128
>512
128
Discussion
dation vulnerability, and challenging formulation develop-
ment, to name few of them.
Because of their wide-spectrum activity and low-resistance
development propensity, AMPs are perceived as potential
candidates for anti-infective drugs. Despite this, none of the
studied molecules has completed the clinical trials with a pos-
itive outcome thus far. As an example, pexiganan (MSI-78), a
22-residue magainin analog, formulated as a 0.8% cream
(Locilex) have recently failed to pass Phase III clinical trials.
Locilex did not exhibit any superiority against a placebo
cream in treating mild infections of diabetic foot ulcers [25].
There are several reasons for limitation of marketing AMPs,
such as high production cost, enzymatic and chemical degra-
Aurein 1.2 is one of the shortest nature–derived AMPs [26].
Owing to only 13 amino acid residues in its backbone and a fair
antimicrobial activity, aurein 1.2 appears to be an interesting
anti-infective drug candidate. In addition, further modifications
of its amino acid residues might provide an analog with an
increased biological activity and chemical stability.
Up to date, a number of studies describing structure-
activity relationship of aurein 1.2 have been published. Li
et al. found Phe13 residue to be an essential asset for antimi-
crobial activity. In this study, aurein 1.2 and its two analogs
F13W (Phe13 residue substituted with Trp) and F13X (Phe13
Table 5 Percentage of hemolysis
[%] caused by test compounds at
Peptide
256
128
64
32
16
8
4
2
1
different concentrations [μg/mL]
G1A
L2A
4.2
0.3
0.0
0.0
0.0
6.6
0.1
0.1
0.7
16.1
0.0
2.9
12.0
0.0
4.5
0.0
0.0
0.0
1.5
0.0
0.0
3.4
5.5
0.0
0.4
6.5
0.0
0.5
0.0
0.0
0.0
0.3
0.0
0.1
0.1
0.0
0.0
0.1
1.2
0,0
0.4
0.0
0.2
0.0
0.3
0.0
0.3
0.0
0.0
0.1
0.1
0.0
0.4
0.0
0.0
0.4
0.0
0.3
0.2
0.5
0.0
0.0
0.3
0.2
0.0
0.5
0.0
0.0
0.6
0.1
0.3
0.3
0.6
0.1
0.0
0.6
17.4
0.0
0.6
0.1
0.0
0.3
0.1
0.2
0.2
0.4
0.2
0.1
0.6
0.1
0.0
0.3
0.0
0.0
0.0
F3A
0.3
0.0
D4A
I5A
61.0
1.1
23.2
0.0
I6A
0.1
0.1
K7A
K8A
I9A
4.6
3.0
89.6
0.0
49.3
0.0
E11A
S12A
F13A
aurein 1.2
40.2
23.5
0.0
11.9
15.4
0.0
58.8
19.2

Probiotics & Antimicro. Prot.
Table 6 Hydrophobicity
parameter (%ACN) of the
peptides, changes in
hydrophobicity (Δ%ACN),
differences derived from
hydrophobicity coefficients of
amino acid residues–HC, and
self-association tendency param-
Peptide
%ACN
Δ%ACN (analog–aurein 1.2)
ΔHC (alanine–substituted residue)*
Tmax [°C]
G1A
L2A
41.8
38.3
41.1
45.7
39.5
38.2
47.9
48.0
37.8
43.5
45.7
37.8
44.7
− 2.9
− 6.4
− 3.6
+ 1.0
− 5.2
− 6.5
+ 3.2
+ 3.3
− 6.9
− 1.2
+ 1.0
− 6.9
0.0
− 0.15
− 3.44
− 4.74
+ 0.26
− 3.42
− 3.42
+ 1.68
+ 1.68
− 3.42
+ 0.16
+ 0.68
− 4.74
0.0
30
25
25
30
20
20
30
30
15
30
30
25
30
F3A
D4A
I5A
eter (Tmax
)
I6A
K7A
K8A
I9A
E11A
S12A
F13A
aurein 1.2
*Values of hydrophobicity coefficient for the following: Ala (0.06), Asp (− 0.20), Glu (− 0.10), Gly (0.21), Ile (3.48),
Leu (3.50), Lys (− 1.62), Phe (4.80), Ser (− 0.62); derived from analyses performed on RP-HPLC system with
conditions closely similar to those applied in this study (C18 stationary phase, water/ACN with 0.1% TFA) [24]
residue substituted with Phg) were compared regarding their
helicity, hydrophobicity, and antimicrobial activity against
E. coli. Both substitutions led to peptides with a lesser antimi-
crobial activity than that of aurein 1.2. As the studied mole-
cules were characterized by similar helicities, it was suggested
that this decrease in activity resulted from a different location
of aromatic ring of the last residue relative to peptide back-
bone [27]. This suggestion was further explored by Shahmiri
et al.. According to the authors, both phenylalanine residues of
aurein 1.2 play a crucial role in membrane anchoring, with
Phe13 being more influential [28]. Again, Shahmiri et al. rec-
ognized that aurein 1.2 C-terminal amidation directly influ-
ences membrane disruption through promotion of peptide ag-
gregation [29]. Soufian et al. reported increase in antimicrobi-
al activity of aurein 1.2 analog in which N-terminal glycine
was substituted with arginine. They suggest that this augmen-
tation of antimicrobial activity is the result of a higher net
charge [30]. Furthermore, Sajjadiyan et al. assigned an essen-
tial role in lytic activity of aurein 1.2 to Phe3, Lys7, and Lys8.
They suggest that those three amino acid residues play a cru-
cial role in absorbing water molecules into the membrane [31].
In our study, alanine scanning was conducted to establish a
relationship among the structure, antimicrobial activity, and sta-
bility of aurein 1.2. As a result, several interesting observations
were made. Substitution of Asp4 and Glu11 residues resulted in
molecules with enhanced antimicrobial activity against almost
all strains of the microorganisms. Those negatively charged
residues make up the central part of the hydrophilic face of
aurein’s 1.2 α-helix (Fig. 2). Thus, the mutation of each of them
into an alanine residue affected the amphipathicity of the helical
structure and, consequently, produced analogs with a depressed
helicity. Interestingly, this outcome did not affect the antimicro-
bial activity in a negative sense. Moreover, as no significant
difference in Tmax parameter between E11A, D4A, and aurein
1.2 has been found, the increased antimicrobial activity could
also not be related to an enhanced tendency to self-association.
In addition, both analogs exhibited a lower proteolytic stability
than their counterpart (44%, 17%, and 65% of remaining pep-
tide after 6-h incubation in bovine serum, respectively) suggest-
ing that the enhanced antimicrobial activity could not be the
result of extended time of action. The most reliable reason for
this enhancement of the activity is an increased positive net
charge of the analogs. Placing electrically neutral alanine resi-
due in position of aspartate and glutamate residues resulted in a
peptide with a negatively charged microbial membrane interac-
tion enhancement and elimination of electrostatic repulsion.
All analogs with hydrophobic residue (phenylalanine, iso-
leucine, or leucine) substituted with the alanine residue were
Table 7 Percentage of
Peptide
Remaining peptide [%]
peptide remaining after
6 h of incubation at 37 °C
in serum
G1A
L2A
16
6
F3A
38
44
71
29
59
36
6
D4A
I5A
I6A
K7A
K8A
I9A
E11A
S12A
F13A
aurein 1.2
17
77
54
65

Probiotics & Antimicro. Prot.
Table 8 α-Helical content and the Θ222/Θ208 ratio determined by CD.
Deconvolution of the CD spectra was carried out using CDPro software
with CONTINILL algorithm
Peptide
SDS
DPC
Helicity %
Θ222/Θ208
Helicity %
Θ222/Θ208
G1A
L2A
63
84
94
71
72
70
89
76
68
75
76
72
86
0.82
0.84
0.82
0.88
0.90
0.86
0.86
0.87
0.83
0.84
0.83
0.83
0.87
91
89
69
87
79
67
78
89
80
87
74
61
95
0.81
0.83
0.83
0.87
0.89
0.84
0.85
0.85
0.81
0.77
0.79
0.80
0.84
F3A
D4A
I5A
I6A
K7A
K8A
I9A
E11A
S12A
F13A
aurein 1.2
interactions essential for the peptide mechanism of action.
Moreover, it might reduce self-association tendencies, helical
structure stabilization, and anchoring of the peptides into the
microbial bilayers, this ultimately leading to suppression of
the antimicrobial activity. The importance of hydrophobic side
chains in self-association and irrelevance of non-hydrophobic
residues strongly suggest that peptide-peptide attraction relies
on interactions between hydrophobic faces of aurein 1.2. It has
been found that hydrophobicity (retention time) of α-helical
peptides is correlated with peptide self-association ability,
helicity, hemolysis, and antimicrobial activity. Moreover, an-
timicrobial activity versus hydrophobicity has an optimum
window. If it is exceeded, antimicrobial activity will decrease
Fig. 1 Far-CD spectra of aurein 1.2 and its alanine scanning analogs
recorded in a surfactant-free phosphate buffer (PBS), buffered sodium
dodecyl sulfate (SDS), and dodecylphosphocholine (DPC) solutions
characterized by a lower antimicrobial activity than that of
aurein 1.2. This phenomenon might be the result of cumula-
tive influence of the reduced Tmax parameter, hydrophobicity,
and helicity of the analogs. Substitution of hydrophobic side
chains on the non-polar face of the aurein 1.2 α-helix with a
shorter side chain of alanine is likely to affect hydrophobic
Fig. 2 Helical wheel projection of aurein 1.2. Pink color represents acidic
amino acids, blue–basic amino acids, green–polar, and uncharged amino
acid and blank–hydrophobic amino acids

Probiotics & Antimicro. Prot.
[32]. Supplementary Figs. 3–7 present the change in hydro-
phobicity of the peptides (%ACN of analog − %ACN of aurein 1.2
activity and lower hemolysis than K8A. However, antimicro-
bial activity of K8A against E. faecalis, E. coli, and
C. albicans is markedly higher (Supplementary Figs. 4, 5,
and 7, respectively). Interestingly, increasing the net charge
(+ 2) and/or hydrophobicity of aurein 1.2 analogs does not
result in a stronger anticandidal activity. However, reducing
the net charge has a negative effect (Supplementary Fig. 7).
Presumably, Asp4, Glu11, and Ser12 may contribute to in
peptide-yeast cell interaction and this factor overweighs the
impact of the increasing hydrophobicity and net charge of
respective analogs. An opposite effect of substitution of neg-
atively charged residues is evident in the case of eradication of
E. faecalis biofilm—D4A has distinctly lower MBEC than
E11A (Table 3). Interestingly, S12A has a similar activity to
that of D4A which definitely surpasses the parent molecule in
this regard. Those findings indicate that Glu11 can contribute
to disruption of the mature E. faecalis biofilm. However, it is
worth noting that this analog is at the same time more hydro-
philic than aurein 1.2, this could partially diminish the effect
of the increasing net charge. Scanning of the aurein 1.2 pep-
tide library revealed similar tendencies of the most of the
microbial strains in microbiological assays (i.e., D4A, E11A,
and S12A were characterized by enhanced activity, whereas
activities of the remaining peptides were similar or decreased
when compared to aurein 1.2). Nevertheless, a deviation from
this trend was noticed in the case of C. albicans and the mea-
surements of adhesive properties. Furthermore, eradication of
mature structures was observed only for aurein 1.2 and three
other analogs. On the other hand, all the tested compounds were
found to inhibit the early stages of Candida biofilm formation.
This can be explained by the fact that aurein 1.2 and its analogs
can interfere with the expression of genes involved in the in-
duction of hyphae formation, adhesion, and extracellular matrix
production such as Ras1-cAMP-Efg1 and MAP kinase
[33–35]. However, this statement should be supported by ap-
propriate analyses of gene expression. Another hypothesis
claims that the test compounds could bind to carbohydrates
making up part of C. albicans cell wall and which particularly
take part in adhesion [36]. However, Lorenzón et al. in their
study on aurein 1.2 and mannan interactions do not support the
hypothesis that aurein 1.2 interacts with the main carbohydrate
of the yeast cell wall [37]. On the other hand, the dimeric analog
of aurein 1.2 effectively binds to mannan and aggregate
C. albicans [13]. Peptide analogs used in this study were inef-
fective against yeast biofilm. Only the parent molecule and
S12A exhibited noticeable antibiofilm activity.
)
versus change in MIC (log2X, where X is a quotient of the
MIC of an analog by the MIC of aurein 1.2). Importantly, a
value of 1024 was applied for calculations when MIC was >
512 μg/mL. With S. aureus, MIC apparently depends on the
peptide hydrophobicity and the net charge. K7A and K8A are
more hydrophobic than aurein 1.2, but their net charge is
lower. The effect of reduced net charge overweighs the in-
creased hydrophobicity, this leading to a reduced antimicrobi-
al activity of these analogs. The increased net charge also
overweighs effect of reduced hydrophobicity. As a result,
E11A has a higher antistaphylococcal activity than the parent
molecule. The strongest antimicrobial analog (D4A) has both
the higher hydrophobicity and net charge than aurein 1.2.
Moreover, substitution of Ser12 also resulted in an analog
with a higher hydrophobicity and antistaphylococcal activity.
With other microorganisms, S12Awas equally active or a less
active against P. aeruginosa than the parent molecule. Similar
to D4A and E11A, S12A was characterized by an equal Tmax
parameter as compared to that of aurein 1.2. However, in
contrast to those analogs, the net charge of S12A equals to
that of aurein 1.2. Taking into account the fact that S12A
retained 77% of initial concentration after 6 h incubation in
bovine serum solution (37 °C), the increased antimicrobial
activity, as compared to that of aurein 1.2, could be linked
with its increased stability. However, as bovine serum might
significantly differ from microbial culture medium regarding
enzyme composition, further comparative studies are neces-
sary. Moreover, it seems that the serine residue interacts with
bacterial membrane components, this being vital for mem-
brane disruption. This effect is irrelevant with S. aureus, but
essential for P. aeruginosa cell surface-peptide interaction
(Supplementary Fig. 6). Surprisingly, although both K7A
and K8A peptides were characterized by identical nearest-
neighbor effect of substituted lysine residue, net charge,
Tmax values, and similar hydrophobicities (%ACN), they ex-
hibited considerable differences in biological activity. While
K8A showed depressed antimicrobial and increased hemolyt-
ic activities as those of aurein 1.2, K7Awas almost completely
depleted of biological activity. Those differences can be due to
unequal α–helical contents in the SDS and DPC micelles.
Substitution of Lys8 residue with alanine led to a molecule
with a higher α–helical content in DPC micelles (mimicking
mammalian membranes) and lower in SDS micelles (mimick-
ing bacterial membranes) as compared to that of the K7A
analog. In general, K7A adopts a more helical structure in
SDS (89%) than in DPC (78%), and helicity of K8A is higher
in DPC (89%) than in SDS (76%). Furthermore, only the
helicity of K7A in SDS is higher than that of aurein 1.2
(86%). Hypothetically, if the helicity is a critical factor for
antimicrobial activity, and the hydrophobicity remains similar,
it should be found that K7A has a stronger antimicrobial
Hemolysis of the analogs at 256 μg/mL is shown in
Supplementary Fig. 8. It is noteworthy that all the peptides that
are more hydrophilic than aurein 1.2 are simultaneously less
hemolytic. Furthermore, analogs that differ from the parent
molecule in hydrophobicity by more than − 3.6 %ACN evoke
little or no hemolysis. Five peptides were included in a simple
linear regression to visualize hypothetical curve of hemolysis

Probiotics & Antimicro. Prot.
vs hydrophobicity (G1A, F3A, K8A, aurein 1.2, and E11A;
R2 = 0.967), but compounds of this group carry different net
charges. Importantly, D4A, K7A, and S12A are outside of the
hypothesized linearity. In fact, D4A and S12A similar to K7A
exhibited a lower helicity in DPC than did aurein 1.2 (87% and
74%, respectively). Probably, Asp4 and Ser12 interact with
RBC membrane components like zwitterionic phospholipidic
headgroups and thus substitution with alanine leads to lower
hemolysis. Both lysine residues have identical nearest neigh-
bors, isoleucine, and lysine, and thus the difference in K7A and
K8A biological activity results presumably from interactions
with more distant residues. In fact, aurein 1.2 is capable to form
five salt bridges between N-terminal amino group or side chain
of the lysine residue and side chain of aspartic or glutamic acid
(distance i + i + 3/i + 4; Table 9). With E11A, the measured
change in hydrophobicity (the analog is more hydrophilic) is
in contrast to predictions derived from hydrophobicity coeffi-
cients (Table 6, Supplementary Fig. 1). This may be due to the
phenomenon of charge compensation between the carboxyl
and amine groups, i.e., zwitterionic alpha amino acids are more
hydrophobic than could be predicted from the occurrence of
two charged moieties [38]. Cheraghi et al. in molecular dynam-
ics study of aurein 1.2 (5-peptide channel, with hydrophilic
sides of peptides directed towards the center of pentagon
channel) have shown that only in one case (in one chain,
Lys8-Asp4) the residues are far enough to be able to neutralize
each other [39]. Furthermore, it is possible that Asp4 is neu-
tralized with the N-terminal amino group (i, i + 3); hence,
Asp4 can interact less effectively with Lys7 and Lys8 (i,i + 3
and i + 4, respectively). The previous studies on amphipathic
α-helical cationic antimicrobial peptides indicate that an in-
crease in net charge and hydrophobicity leads to stronger anti-
microbial activity and hemolysis, but when the positively
charged amino acid is located on the non-polar face it can
reduce toxic effect [40]. Obviously, Lys8 is not located on
the non-polar face of aurein 1.2 (Fig. 2); nevertheless, the effect
of its substitution with alanine resulted in a compound with the
highest hemolytic activity. Substitution of Lys7 residue (in the
vicinity of peptide polar/non-polar face) resulted in a peptide
with a markedly reduced biological activity. In conclusion,
K8A is more hemolytic due to enhanced hydrophobicity and
K7A is less hemolytic due to the reduction of the net charge
that outweighs the influence of enhanced hydrophobicity. In
other words, effective charge of Lys7 predominates effective
charge of Lys8 due to compensation effect. Further consider-
ations lead to the following hypothesis: the side chain of Glu11
partially compensates positive charge of the Lys8 (i,i + 3) side
chain and the N-terminal amino group partially compensates
Asp4. In effect, Lys7 interacts more efficiently with biological
membranes than does Lys8. Our study revealed that substitu-
tion of Lys7 and Lys8 with alanine resulted in identical anti-
microbial activities against S. aureus and P. aeruginosa (over
the range of 1–512 μg/mL), as opposed to other microbial
strains and RBCs. Nonetheless, previous studies on aurein
1.2 have shown that hydrophobic interactions play a key role
in helicity of peptide bound to micelles and salt bridges are
irrelevant and their existence is uncertain [41].
Individual hydrophobicity of alanine residue is higher than
that of glycine. Nevertheless, substitution of the former resi-
due with alanine, unexpectedly led to G1A—an analog char-
acterized by a lesser hydrophobicity than that of aurein 1.2. In
addition, a significant difference between helical content in
SDS and DPC micelles was noticed. α-Helical content of
G1A was by ca. 25% smaller in SDS micelles than in DPC.
Bearing in mind that SDS micelles, as well as bacterial mem-
branes, are negatively charged, it suggests an essential role
played by glycine residue in microbial selectivity. However,
as a decrease was found in both antimicrobial and hemolytic
activities, evidently, glycine residue in the case of aurein 1.2
contributes to membrane interaction in general.
In summary, helicity, hydrophobicity, and aggregation ten-
dencies play an essential role in microbial specificity of
AMPs. Generally, increase in those values to definite extent
results in enhancement of antimicrobial activity. Further aug-
mentation, however, not only reduces antimicrobial activity
due to peptide aggregation in solution, but also strengthens
hemolytic activity of the molecule [32, 42, 43]. Similar ten-
dencies emerged in the case of studied alanine scan analogs.
Aurein 1.2 is characterized by the highest helicity, Tmax, pa-
rameter, and significant hydrophobicity, at the same time ex-
hibited one of the highest hemolytic activities among the com-
ponents of the peptide library. While observations made for
D4A were comparable to those of aurein 1.2, substitution of
glutamic acid residue with alanine resulted in a molecule
(E11A) with a slightly lower hydrophobicity and, in conse-
quence, in hemolytic activity. On the other hand, a consider-
able decrease in α-helical content, as seen in S12A, also re-
sulted in reduction of hemolysis, while retaining the antimi-
crobial activity. Along with their antimicrobial activity, all
analogs with hydrophobic amino acid residues (phenylala-
nine, isoleucine, or leucine) substituted with alanine exhibited
a huge drop in hemolytic activity, again, as a result of the
decrease in hydrophobicity, Tmax, and helicity.
Table 9 Distances of salt bridges between N-terminal amino group or
side chain of lysine residues and side chain of aspartic or glutamic acid
Asp4
Glu11
N-terminus
Lys7
i,i + 3
i,i + 3
i,i + 4
Irrelevant
i,i + 4
An article with part of experimental section titled BSite-
directed mutagenesis of aurein 1.2^ has been published so
far [44]. However, in fact, the object of alanine scan
Lys8
i,i + 3

Probiotics & Antimicro. Prot.
described there is no aurein 1.2 peptide itself, but a con-
jugate of aurein 1.2 with + 36 GFP-Cre recombinase fu-
sion protein. Even so, our study is the first one in which
the influence of each amino acid residue substitution on
aurein 1.2 antimicrobial, antibiofilm, and hemolytic activ-
ities, secondary structure, hydrophobicity, aggregation
tendencies, and serum stability have been comprehensive-
ly characterized. The correlations established between
those characteristics led to selection of key residues es-
sential for peptide activity. Moreover, residues which did
not affect at all, or even negatively affected biological
activity of aurein 1.2, were also appointed. Those out-
comes can assist in aurein 1.2-based molecule design op-
timization. For instance, peptides with increased antimi-
crobial activity may be obtained by substitution of both
Asp4 and Glu11 with neutral or positively charged resi-
dues. On the other hand, studies on substitution of Ser12
might result in antimicrobial compounds with selectivity
against S. aureus. Additionally, other modifications such
as dimerization [45], hydrocarbon stapling [46], or
lipidation [47] can be also applied to improve pharmaco-
logical properties of aurein 1.2.
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