Search for new aggregable fragments of human insulin

Article abstract of DOI:10.3390/molecules24081600

In this study, three independent methods were used to identify short fragment of both chains of human insulin which are prone for aggregation. In addition, circular dichroism (CD) research was conducted to understand the progress of aggregation over time.

Full text of DOI:10.3390/molecules24081600

molecules
Article
Search for New Aggregable Fragments of
Human Insulin
Monika Swiontek 1, Justyna Fraczyk 1, Joanna Wasko 1, Agata Chaberska 1, Lukasz Pietrzak 2,
Zbigniew J. Kaminski 1 , Lukasz Szymanski 2 , Slawomir Wiak 2 and Beata Kolesinska 1,
*
1
Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland;
monika.swiontek@gmail.com (M.S.); justyna.fraczyk@p.lodz.pl (J.F.); joanna.wasko@p.lodz.pl (J.W.);
agata.chaberska@p.lodz.pl (A.C.); zbigniew.kaminski@p.lodz.pl (Z.J.K.)
Institute of Mechatronics and Information Systems, Faculty of Electrical, Electronic, Computer and Control
Engineering, Lodz University of Technology, Stefanowskiego 18/22, 90-924 Lodz, Poland;
lukasz.pietrzak@p.lodz.pl (L.P.); lukasz.szymanski@p.lodz.pl (L.S.); slawomir.wiak@p.lodz.pl (S.W.)
Correspondence: beata.kolesinska@p.lodz.pl; Tel.: +48-42-631-3149
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 March 2019; Accepted: 20 April 2019; Published: 23 April 2019
Abstract: In this study, three independent methods were used to identify short fragment
of both chains of human insulin which are prone for aggregation. In addition, circular
dichroism (CD) research was conducted to understand the progress of aggregation over time.
The insulin fragments (deca- and pepta-peptides) were obtained by solid-phase synthesis using
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) as a
coupling reagent. Systematic studies allowed identification of the new fragments, expected to be engaged
in triggering aggregation of the entire structure of human insulin under physiological conditions. It was
found that the aggregation process occurs through various structural conformers and may favor the
formation of a fibrous structure of aggregate.
Keywords: Aggregation; amyloid deposits; amyloid-like fiber formation; diabetes; SPPS; triazine
coupling reagents
1. Introduction
Insulin is involved in many bodily processes. It participates in DNA replication and protein
synthesis by regulating amino acid uptake and modulating the activity of many enzymes, such as
hexokinase, phosphofructokinase, glycogen synthase and glycogen phosphorylase [1]. Insulin is also
involved in the biosynthesis of glycogen and its storage in the liver, resulting in lowering of blood
glucose. It accelerates the biosynthesis of fats and slows down proteolysis, lipolysis and glucogenesis.
It prevents glucose synthesis from non-sugar substrates. Impairment of its overriding functions,
through its destruction by autoimmune
is correlated with the development of type I and type II diabetes [2,3
β
cells or loss of biological activity, causes hyperglycemia and
]. Human insulin, with a molecular
weight of 5808 Da, is formed from 51 amino acid residues. It is built from two chains: Chain A (alpha),
containing 21 amino acid residues and chain B (beta), composed of 30 amino acid residues. Both chains
are connected by two disulfide bridges formed between cysteines A7–B7 and A20–B19. In chain A
there is a third disulfide bridge connecting the two cysteines in position A6–A11. Insulin aggregation
is a crucial factor in the development of diabetes [4–7]. Aggregation of insulin in solution is also an
important technological problem for the production, storage and use of insulin preparations [8–10].
The ability of a protein to aggregate depends on its primary structure. The most important
factors contributing to aggregation are the hydrophobicity of amino acid residues, the nature of the
amino acid side chains, the balance between polar and non-polar amino acids and the presence of
Molecules 2019, 24, 1600; doi:10.3390/molecules24081600
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aromatic ring interactions. Many differently structured proteins and polypeptides are affected by
inappropriate folding, resulting in the formation of stable and insoluble peptide/protein structures.
Altered spatial structures result in the formation of amyloid deposits, leading directly to the development
of conformational diseases or amyloidosis [
of amyloid fibers consisting of characteristic structured
environmental conditions and is accelerated by high temperature and low pH [
1
,
2
]. In the case of insulin, it is postulated that the formation
-sheets occurs under a wide range of
11]. A characteristic
β
3,
feature of amyloidogenic proteins is the presence in their structures of short fragments responsible for
initiating aggregation, which are also involved in the stabilization of amyloid fibrils. These fragments,
called hot spots, form amyloidogenic cores and can contain up to three or four amino acid residues.
In vitro, these undergo aggregation, leading to amyloid-like structures [10,12–17].
The first atomic-level view of the interactions between segments of insulin, which may form part of
a fibrillar spine (“amyloidogenic core”), revealed single crystal structures in the fibril-forming peptide
segments A13–A19 H–LYQLENY–OH, A13–A18 H–LYQLEN–OH and B12–B17 H–VEALYL–OH.
Biophysical studies suggest that the B chain of insulin, or a segment of it, may be the preliminary
determinant of insulin fibrillation. The amyloidogenic core of insulin consists of two antiparallel
arranged B12–B17 fragments, which due to their specific mutual orientation form a “steric zip”,
which is stabilized by hydrophobic interactions between aliphatic amino acid residues, hydrogen bonds
between insulin chains and particularly by the interaction of hydroxyl groups in the tyrosine–tyrosine
side chains. This structure interacts with the A13–A19 LYQLENY fragment of chain A, which is
involved in the stabilization of the β-structure of the amyloidogenic core [18,19]. At the periphery of
the insulin fibrillar spine model, the N- and C-termini retain the native-like structure of the insulin
molecule. A similar model, with a “steric zipper” spine and native-like structure on the periphery, was
proposed for the designed amyloid of ribonuclease A [20]. These findings were confirmed by Eisenberg,
who classified the fibrillary spine of insulin within the face-to-back class of “steric zippers” [19] formed
by B12–17 VEALYL, B11–16 LVEALY and A13–18 LYQLEN. These short peptides have been described
and characterized by Congo Red (CR), Thioflavin T (ThT), electron microscopy and X-ray diffraction
assays. In the literature there is a plenty of examples of peptides as short as three amino acid residues,
which form fibrils and which then play a role in aggregation of the whole protein [10,12–17]. Results of
this studies has been used by Masunov et al. for Molecular Dynamics (MD) studies on full-length
insulin amyloid oligomer models [21]. More recently another group also performed MD simulation.
Studies of molecular dynamics indicated strong interaction between the docked fragment LVEALYL
and fragment B22–B27 RGFFYT. In the best model, RGFFYT was bound to insulin by one hydrogen
bond with Phe–B25. It has also been found that the binding of LVEALYL to RGFFYT is driven by
electrostatic interaction, because both the ligand and receptor expose charged amino acid residues.
Using MD simulations and the Zyggregator method to calculate the propensity profile, it has been
predicted that RGFFYT can also self-assemble. It was postulated that presence of VE (fragment 2–3) CS
(fragment (11–12) from chain A and FVNQH (fragment 1–5) and RGFF (fragment 22–25) is conducive
to the increase of β-strand content [22]. This data were assigned to insulin, but also to a mixture of
insulin and fragment LVEALYL (hot spot of insulin). Authors have indicated especially on fragment
B22–27 RGFFYT, which interacts with B12–17 LVEALYL and hypothesized that this fragment is prone
to aggregation, which when coming back to experimental data was quite surprising, since neither this
region nor even neighboring ones were described as amyloidogenic.
This was our inspiration to check this hypothesis by experiments under physiological conditions
(pH = 7.2, 37 ◦C). Therefore, the main aim of the present study was to conduct a systematic search for
new insulin fragments prone to aggregation. Finding aggregating fragments should assist the rational
design of hormone aggregation inhibitors, facilitating the search for new drugs for the treatment of
diabetes. Short fragments covering the complete structure of human insulin were synthesized and their
propensity to aggregate studied, in order to select new aggregating peptides than those previously
identified. The sequences of all the fragments of insulin designed for testing are presented in Table 1.

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Table 1. Designed fragments of human insulin covering the entire hormone structure.
Chain A
Chain B
A1–A10 H–GIVEQCCTSI–OH (1)
A1–A5 H–GIVEQ–OH (2)
B1–B10 H–FVNQHLCGSH–OH (7)
B1–B5 H–FVNQH–OH (8)
A6–A10 H–CCTSI–OH (3)
B6–B10 H–LCGSH–OH (9)
A11–A21 H–CSLYQLENYCN–OH (4)
A11–A16 H–CSLYQL–OH (5)
A17–A21 H–ENYCN–OH (6)
B11–B20 H–LVEALYLVCG–OH (10)
B11–B15 H–LVEAL–OH (11)
B16–B20 H–YLVCG–OH (12)
B21–B30 H–ERGFFYTPKT–OH (13)
B21–B25 H–ERGFF–OH (14)
B26–B30 H–YTPKT–OH (15)
As a positive control in the studies, we used fragments of insulin already described in the
literature as having the ability to form aggregates: A13–A19 (H–LYQLENY–OH) (16) and B12–B17
(H–VEALYL–OH) (17). Fragment B22–B27 (H–RGFFYT–OH) (18), which is suspected of having
aggregable properties, was also used [22].
2. Results and Discussion
Peptides 1–18 were synthesized according to the solid phase peptide synthesis (SPPS) method using
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-)
as a coupling reagent [23]. The purity of the crude peptides increased from 72–97% to 96–99%
after purification by HPLC. The starting point for the studies was the aggregation of already known
insulin-inscribed amyloidogenic cores. Two known aggregating fragments derived from A and B chains
A13–A19 (H–LYQLENY–OH) (16) and B12–B17 (H–VEALYL–OH) (17) were used. Also, fragment
B22–B27 (H–RGFFYT–OH) (18), which is suspected of having this property, was investigated.
In addition, human insulin was used in our studies as a reference point. The aggregation process was
carried out under physiological conditions: pH, 7.2; temperature, 37 ◦C. Such mild conditions were
selected to mimic, as far as possible, the natural conditions inside the human body.
The result of the aggregation process is the formation of spatially ordered peptide structures
driven by weak interactions between amino-acid residues. It was therefore decided that susceptibility
to aggregation would be examined by three independent methods, nonspecific for amyloids,
but recommended [24–32] for monitoring protein/peptide aggregation. The following tests were
used: The Congo Red test, the Thioflavin T assay, and microscopic examination of samples stained
with Congo Red. At this stage of the research, using three independent, nonspecific assays did not
allow outright differentiation of amyloid fiber from aggregates, but we expected that it would facilitate
selection of fragments that are susceptible to aggregation. Based on these independent methods,
recommended [24–32] for monitoring protein/peptide aggregation, it was found that both B12–B17
and A13–A19 fragments had attributes characteristic of aggregable peptides under physiological
conditions. For both insulin aggregating fragments (fragments B12–B17 and A13–A19) known from the
literature [18,19], a characteristic decrease in absorbance was observed over time, with a simultaneous
shift in the maximum absorbance from 489 nm to 542.5 nm (Figure 1a). In the case of the fragment
suspected of aggregation ability, B22–B27, on the first day of aggregation the Congo Red (CR) test
showed an increase in absorbance compared to CR with the simultaneous appearance of a new
absorbance maximum at 542.5 nm. Measurements on subsequent days of incubation showed a decrease
in absorbance, while maintaining the characteristic shift of the maximum absorbance to around 550 nm.

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Figure 1. Cont.

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Figure 1.
B22–B27 H–RGFFYT–OH (18) in the presence of Congo Red (CR). Spectra show results obtained on the
1st, 2nd, 3rd and 4th days of incubation; ( ) fluorescence intensity spectra of peptides 16 17 and 18 in
the presence of Thioflavin T (ThT), wavelength = 485 nm, 4th day of incubation; ( ) pictures of 16 17
and 18, without polarized filter (left side), with polarized filter (right side). Scale bars, 10 m. In all
cases, samples were taken for microscopic examination on the 4th day of incubation; ( ) UV spectra of
(a) UV spectra of A13–A19 H–LYQLENY–OH) (16), B12–B17 H–VEALYL–OH (17) and
b
,
c
,
µ
d
human insulin in the presence of CR (spectra registered on the 1st, 2nd, 3rd and 4th days of incubation),
fluorescence intensity spectra of human insulin in the presence of ThT (spectra registered on the 1st,
2nd, 3rd and 4th days of incubation) and pictures of human insulin without polarized filter (left side),
with polarized filter (right side).
The CR test thus confirmed that the fragment B22–B27 suspected of aggregation properties could
be another aggregable human insulin core. The fluorescence assay with Thioflavin T (ThT) also
confirmed the aggregable properties of B12–B17 and A13–A19 insulin fragments. On the spectra,
a characteristic increase in fluorescence intensity was observed from 7.8
Thioflavin T) to 6.9
106 for H–LYQLENY–OH (Figure 1b). However, when the increases in the
intensity of fluorescence from insulin fragments A13–A19 and B12–B17 were compared, it was noticed
that fluorescence intensity increased from 5.6
106 to 6.9 106 for H–LYQLENY–OH and from
×
105 (for pure incubated
×
×
×
2.8 × 106 to 3 × 106 for H–VEALYL–OH. It is surprising that the increase in fluorescence intensity was
lower for the B12–B17 fragment, with documented aggregating properties, than in the case of the
B22–B27 fragment, which is only suspected of only having the ability to form aggregates. The results
of tests with human insulin indicate that applied, nonspecific tests can be used to search for new
fragments with aggregation properties (Figure 1d). In the microscope test, characteristic fibrous
structures were observed in both polarized and non-polarized light. The result of the UV assay with
CR was also unequivocal. On the UV spectra the characteristic shift of the maximum absorbance and
the decrease in absorbance during incubation was observed. Surprising was the result of the ThT
assay, where the observed fluorescence intensity value 4.85
×
106 was lower than the data obtained
for A13–A19 H–LYQLENY–OH) (16). However, the positive result of two of the three independent,
nonspecific tests allows the confirmation of aggregation properties of human insulin. Thus, the use of
nonspecific methods to study the known aggregable insulin fragments and those suspected of this
property, as well as human insulin, has let us use the results of these tests as a benchmark in testing
new fragments of insulin, to select regions of the hormone prone to aggregation, which may be the
basis for further studies used specifically for amyloid tests.

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In the next stage of the study, the same tests were carried out to examine the susceptibility to
aggregation of peptides, which are fragments of the A-chain of insulin (Figure 2). Based on the results
of the CR test, all peptides lowered absorbance in the presence of the dye. The most spectacular
decrease in absorbance was observed in the cases of peptides and . All the fragments derived from
human insulin chain A showed a characteristic shift of maximum absorption, from 489 nm to 545 nm
(Figure 2a). Analysis of the results of the ThT test also confirms the ability of peptides to aggregate and
1–6
1–6
5
6
1–6
form aggregates and fibrous structures. In each case, a significant increase in the fluorescence intensity of
the dye was observed in the presence of the tested peptides compared to the control, for which Thioflavin
T alone was used (Figure 2b). The fluorescence intensity values for deca-peptides
for the shorter fragments and were lower. These results are extremely interesting, particularly as
regards peptide , because microscopic studies of this peptide indicated the presence of mainly amorphous
1 and 4 were higher than
2,3
5,6
1
structures, with a small proportion of fibrous structures (Figure 2c).
Figure 2. Cont.

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Figure 2.
H–CSLYQLENYCN–OH (
Spectra were obtained on the 4th day of incubation; (
presence of ThT, wavelength = 485 nm, 4th day of incubation; (
of , without polarized filter (left side), with polarized filter (right side). Scale bars, 10
cases, samples were taken for microscopic examination on the 4th day of incubation.
(
a
) UV spectra of H–GIVEQCCTSI–OH (
), H–CSLYQL–OH ( ) and H–ENYCN–OH (
) fluorescence intensity of
) pictures of peptides; (d
1
), H–GIVEQ–OH (
) in the presence of CR.
peptides in the
) pictures
m. In all
2), H–CCTSI–OH (3),
4
5
6
b
1–6
c
1
–
3
4–
6
µ
Microscopic studies of the remaining fragments derived from the A-chain of human insulin
revealed that characteristic fibrous structures had formed during aggregation in the presence of CR.
Fibrous structures were visible both with and without a polarizing filter (Figure 2c,d).
In the next stage of the research, the same tests were made on the ability of large and small
fragments derived from the B chain of human insulin (peptides 7–15) to form aggregates containing
fibrous structures (Figure 3). The CR test showed that, for most fragments of the B-chain of human
insulin, characteristic decreases in absorbance occurred and the maximum absorption shifted from
489 nm to 545 nm (Figure 3a), indicating the susceptibility of these fragments to aggregation. Only for
peptide 13 was a much higher absorbance compared to CR observed accompanied by the maximum
absorbance shift almost to 550 nm. Moreover, the shape of the UV–Vis spectra of peptides 10 and 12
was quite unexpected, because in both cases the spectra were almost flat. Thus, the nonspecific Congo
Red assay does not definitely answer which fragments of B chain of insulin are aggregable ones.
The ThT assay showed for all fragments of B chain (peptides 7–15) a higher fluorescence intensity
than the control (Figure 3b). The highest intensity of fluorescence was observed for deca-peptides
10 and 12 as well as for penta-peptide 13. High fluorescence intensity indicates the ability to form
aggregates. This result is also consistent with the data obtained from microscopic examination.
The peptides 10
,
12 and 13 formed fibrous structures visible both with and without a polarizing filter
and 15, suggesting the lowest
(Figure 3d,e). The lowest fluorescence intensity was seen for peptides
7
potential for aggregation. This result is consistent with the data from microscopic studies, in which
mainly amorphous structures were observed, contaminated only with fibrous structures. The results
presented in Figures 2 and 3 confirm that, with structures formed as a result of weak interactions,
the use of a single analytical method may lead to erroneous conclusions. A summary of the results of
CR, ThT and microscopic tests for known aggregable fragments of insulin and tested peptides
presented in Table 2.
1–15 is

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Figure 3. Cont.

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Figure 3.
(a) UV spectra of H–FVNQHLCGSH–OH (7), H–FVNQH–OH (8), H–LCGSH–OH (9),
H–LVEALYLVCG–OH (10), H–LVEAL–OH (11), H–YLVCG–OH (12), H–ERGFFYTPKT–OH (13),
H–ERGFF–OH (14) and H–YTPKT–OH (15) in the presence of CR. Spectra were obtained on the 4th
day of incubation; (
nm, 4th day of incubation; (
13 15 peptides, without polarized filter (left side), with polarized filter (right side). Scale bars, 10
In all cases, samples were taken for microscopic examination on the 4th day of incubation.
b
) fluorescence intensity of
7
–
15 peptides in the presence of ThT, wavelength = 485
peptides; ( ) pictures of 10 12 peptides; ( ) pictures of
m.
c) pictures of
7–
9
d
–
e
–
µ
The assay results indicate that among the tested peptides 1–15, it was not possible to indicate
a fragment that in all three tests would give a very good result as in the case of peptide A13–A19
H–LYQLENY–OH (16). It was assumed that the positive result of the two independent tests predisposed
the examined fragments of insulin to be classified into a group conducive to the aggregation of
the hormone.
Selected peptides 10, 11 and 12, were further examined using an atomic force microscopy
(AFM) microscope. The presence of fibrous structures was observed with a tendency to form larger
agglomerates (Figure 4).
Research using the AFM technique confirmed the fact that structures in the form of fibrils
aggregated with a dominant amorphous phase, which was clearly visible in the case of the peptide
10. The presence of fibrillar structures were observed in sample 12, as a major agglomeration of
particles. In the case of peptide 11, a lamellar structure characteristic of the crystalline phase of partially
crystalline polymers was seen, presumably indicating the possibility of homogeneous crystallization.

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However, spherulites were not clearly defined. Weak interactions were most likely responsible for
form of organization.
Table 2. Susceptibility to aggregation of peptides 1–15.
Microscopic Examination,
Morphology
Known Hot Spots of Insulin or Fragment Suspected of Having Amyloidogenic Properties
Insulin Fragments
CR Assay
ThT Assay
A13–A19 H–LYQLENY–OH (16)
B12–B17 H–VEALYL–OH (17)
++
++
++
+
++
++
+
B22–B27 H–RGFFYT–OH (18)
++
+
fibrous structure
Chain A
+/-
A1–A10 H–GIVEQCCTSI–OH (1)
+
++
cluster of fibrous and
amorphous structures
+
A1–A5 H–GIVEQ–OH (2)
A6–A10 H–CCTSI–OH (3)
+
+
+
+
+
fibrous structure
+
fibrous structure
+
fibrous structure
+
fibrous structure
+/-
cluster of fibrous and
amorphous structures
A11–A21 H–CSLYQLENYCN–OH (
A11–A16 H–CSLYQL–OH (5)
4
)
++
+
+/-
almost flat spectrum
+/-
A17–A21 H–ENYCN–OH (6)
+
almost flat spectrum
Chain B
+
B1–B10 H–FVNQHLCGSH–OH (7)
B1–B5 H–FVNQH–OH (8)
+
+/-
fibrous structure
+/-
cluster of fibrous and
amorphous structures
+/-
cluster of fibrous and
amorphous structures
+
fibrous structure
+
fibrous structure
+/-
cluster of fibrous and
amorphous structures
+
+
+
B6–B10 H–LCGSH–OH (9)
+/-
+/-
B11–B20 H–LVEALYLVCG–OH (10)
B11–B15 H–LVEAL–OH (11)
+
+
almost flat spectrum
+
+/-
B16–B20 H–YLVCG–OH (12)
+
+
almost flat spectrum
+/-
an absorbance higher
than CR, a characteristic
shift of the maximum
absorbance
+/-
B21–B30 H–ERGFFYTPKT–OH (13)
cluster of fibrous and
amorphous structures
+
B21–B25 H–ERGFF–OH (14)
B26–B30 H–YTPKT–OH (15)
+
+
fibrous structure
+/-
cluster of fibrous and
amorphous structures
+
+/-
++ very high test result, + positive test result, +/- the result of the test is ambiguous.
Having demonstrated the ability of several A and B chain fragments to form fibrous structures,
studies were continued to analyze the secondary spatial structures formed by fragments of insulin
incubated in phosphate buffer solution, pH 7.2, at 0.1 mg/mL concentration at room temperature. For this
purpose, selected fragments of human insulin were tested using circular dichroism (CD) spectroscopy.
Interpretation of the CD spectra was challenging. The general rule states that peptides with a
random coil structure have a minimum at 195–200 nm. Peptides rich in
at 216 nm. Above that, the progress of aggregation process is accompanied by a shift of the minima to
longer wavelengths. This interpretation has been used previously for -amyloid [33 35]. All such
β-structures have a minimum
β
–

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shifts refer to situations in which random coil conformation changed via the
α
-helix, which only
in subsequent steps rearranged to amyloid fibrils. Dunstan et al. also describe protofilaments in
the preliminary phase of the aggregation of amorphous aggregates as initially containing mainly
structures [36], thus the -helix structure is postulated on the way/pathway to mature amyloid fibrils.
This is what we too have concluded from our research [16].
α
α
Due to the poor predictability of this multistage conformational transformation, it was essential to
combine different analytic methods to determine whether we were dealing with amorphous aggregates,
protofibrils or with mature fibrils. Only mature fibrils contain only
interpretation of CD spectra can be unreliable, especially when it comes to
often mistaken for -helix rich structures. There is also a point of structural diversity, which is assigned
to -structures. This diversity refers to the orientation of the peptide backbone and the direction of its
twist. Both effects have an influence on the observed spectra. The following configurations should be
mentioned: Anti-1 left-twisted -strand (left-hand twisted antiparallel -sheet), anti-2 relaxed -strand
(relaxed, slightly right-hand twisted antiparallel -sheet), anti-3 right-twisted -strand (right-hand
twisted antiparallel -sheet) and parallel -strand (parallel -sheet). All of these are very similar in
shape to the distorted -helix and pose similar minima at 195–200 nm [37 38]. For instance, the anti-1
β-sheet structures. Moreover,
β-structures, which are very
α
β
β
β
β
β
β
β
β
β
α
,
structure has two characteristic minima at 190–195 nm and 225–230 nm. The anti-2 structure gives one
minimum at 180–190 nm and two small minima at 215–220 nm and 235–240 nm, at the very end of the
spectrum. Anti-3 also possesses a minimum in the range of 190–200 nm, but also two small maxima in
the ranges of 205–210 and 230–235. Finally, the parallel
one small minimum at 210–220 nm.
β-strand has one maximum at 200–210 nm and
Figure 4. Cont.

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Figure 4. Three-dimensional structures of selected peptides, atomic force microscopy images.
We first looked for characteristic changes on the CD spectra of the insulin A chain. A characteristic
shift of the minimum from 195–200 nm to 205 nm after five days of incubation was observed for
deca-peptide
starting from a random coil structure to a more
our microscopic measurements, in which clusters of mainly fibrous and amorphous structures were
observed. The same effect was found for penta-peptide (A6–A10 fragment). This shift of the
minimum from 195 nm to 215 nm could indicate an anti-3 right twisted -strand. It is also worth
mentioning that microscopic studies also revealed fibrous structures for this peptide. For penta-peptide
(fragment A1–A5), changes were noted in the spectra. After two days of incubation there was a
1
(A1–A10 fragment). This effect could indicate structural changes in the peptide,
β-rich structure. These results are supported by
3
β
2
random coil structure, but after three more days of incubation the CD spectrum began changing.
The shape of spectra became irregular and it was not easy to assign a definite conformation (Figure 5a–c).
Nevertheless, a fibrous structure was observed under the microscope. Very interesting spectra were
also recorded for deca-peptide
4 (A11–A21 fragment), strongly suggesting the presence of structure
β, which could be a mixture of anti-1 and anti-3 structures (Figure 5d). It is worth emphasizing that
this peptide contains the shorter A13–A19 fragment, which has been described in the literature as an
amyloidogenic core of insulin. Again, fibers were seen under the microscope.
Turning to chain B of insulin, we observed changes in some regions of the spectra, which could
indicate the presence of structure β. These were visible in three fragments. Deca-peptide 7 (B1–B10
fragment) had a minimum at 195 nm from the very beginning of incubation. However, between the
second and fifth days this minimum shifted to 205 nm and was accompanied by the appearance of
a minimum at 221 nm, which could indicate structure
aggregates. Both penta-peptides and derived from deca-peptide
aggregating propensity. In the case of peptide (B1–B5 fragment), both anti-1 and anti-3 structures
were observed, and for fragment B6–B10 (peptide ) anti-1 and anti-3 structures slowly changed to
β. Microscope measurements again revealed
8
9
7
(B1–B10 fragment) had an
8
9

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relaxed β-strands (Figure 6a–c). Surprisingly, these had never been considered as fragments that
undergo aggregation processes.
Figure 5. Circular dichroism (CD) spectra of fragments (a) A1–A10 (peptide 1), (b) A1–A5 (peptide 2),
(c) A6–A10 (peptide 3), (d) A11–A21 (peptide 4). The peptides were incubated in phosphate buffer
solution, pH 7.2, concentration 0.1 mg/mL at room temperature: 0 day (CD spectrum of peptides
after dissolving the sample); 2 days (CD spectrum of peptides after 2 days of incubation); 5 days (CD
spectrum of peptides after 5 days of incubation).
Very interesting changes in the CD spectra were observed for fragments belonging to deca-peptide
10 (B11–B20 fragment). Deca-peptide 10 has a minima characteristic for an anti-1 structure, but the
shorter B11–B15 fragment belonging to deca-peptide 10 only showed a random coil structure. It is also
very interesting to note that fragment B16–B20 (peptide 12) (Figure 7a–c) behaved in the same way as
fragment B1–B10 (peptide 7), described previously.

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Figure 6. CD spectra of fragments (
a) B1–B10 (peptide 7), (b) B1–B5 (peptide 8), (c) B6–B10 (peptide 9).
Experimental conditions were identical to those described above.
Figure 7. CD spectra of fragments (a) B11–B20 (peptide 10), (b) B11–B15 (peptide 11), (c) B16–B20
(peptide 12). Experimental conditions were identical to those described above.

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These results suggest that the amino acid tyrosine could have an essential role in the aggregation
of fragment B16–B20 (peptide 12). Fragment B12–B17 likewise appears to play an important role in the
aggregation of insulin, as part of a “steric zipper” [19].
Considering molecular dynamics studies [22], which have suggested, that there may be more
short fragments and even amino acid residues affecting the increase of content of β-strand, the results
of our studies can support this hypothesis. We found, that fragments A11–21, A11–16 undergo
aggregation, and they cover the A11–A12 fragment and the A15 residue which are indicated as crucial
for aggregation. Found by us, fragments A1–A10, A1–A5 possess aggregation propensity and both
contain the A3 and A4 residues also influencing aggregation. We have also confirmed, that fragment
B22–B27, and also a shorter one, B21–B25 undergo aggregation processes. These results are not
contradicting with MD simulations results. Moreover, fragment B1–5 with a β-strand conformation
assigned by MD simulations, also showed aggregating propensity according to our experimental data.
3. Materials and Methods
Solid phase peptide synthesis (SPPS) was used to synthesize peptides 1–18 according to the Fmoc
methodology. Fmoc-protected amino acids were purchased from Novabiochem (San Diego, CA, USA)
or Bachem AG (Bubendorf, Switzerland). Human insulin was purchased from Sigma-Aldrich (Saint
Louis, MO, USA).
3.1. Synthesis of Peptides
3.1.1. Loading of the 2-Chlorotrityl Chloride Resin (GP1)
The amino acid (3 equivalents relative to the resin) and EtNiPr2 (6 equivalents relative to
the resin) were dissolved in CH2Cl2 (10 mL/g resin), containing if necessary, a small amount of
N,N-dimethylformamide (DMF) to facilitate dissolution of the amino acid. The 2-chlorotrityl chloride
resin was preswollen in CH2Cl2 for 1 h, and then the solution containing amino acid was added.
The mixture was shaken for 30–120 min, then the resin was washed with CH2Cl2/MeOH/EtNiPr2
17:2:1 (3
×
), DMF (2
×
) and CH2Cl2 (3 ). The functionalized resin was dried in a vacuum desiccator to
×
constant mass. Deprotection of the first amino acid was prolongated (2 × 30 min).
3.1.2. Standard Coupling Procedure (GP 2)
Three equivalents of amino acid and 6 equivalents of N-methylmorpholine (NMM) were mixed and
added to the resin, followed by the addition of 3 equivalents of coupling reagent (DMT/NMM/TosO-).
The solution was added to the resin and shaken for 1–2 h. The completion of the reaction was monitored
using the Kaiser test.
3.1.3. Fmoc- Deprotection (GP 3)
The Fmoc protecting group was removed by placing the resin in a solution of 25% piperidine in
with DMF for 30 min. The completion of the reaction was monitored using the Kaiser test.
3.1.4. Cleavage from the Resin (GP 4)
Peptides (1–18) were cleaved from the resin using a mixture of 95% TFA (2,2,2–trifluoroacetic
acid)/2.5% H2O/2.5% TIS (triisopropylsilane) (ca. 2 mL/0.1 g resin). Cleavage was performing over 3 h,
then the resin was filtered off and the filtrate was evaporated. The residue was treated with Et2O to
precipitate the peptide. The resulting solid was filtered off and washed with Et2O. The crude product
was lyophilized and identified by MS. Its purity was determined by RP-HPLC. All HPLC spectra and
MS spectra are presented in Supplementary Materials).

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3.2. Peptides 1–18 Analysis
Analytical RP-HPLC. Performed on a Waters HPLC system (Waters Corporation, Milford, MA,
USA), using a Kinetex Reversed Phase C18 column (100 4.6 mm). A gradient of 0.1% TFA in H2O (B)
and 0.1% TFA in CH3CN (A), at a flow rate 0.4 mL/min was used with UV detection at 220 and 254 nm.
All HPLC spectra of crude 18 peptides are available in Supplementary Materials: Figures S1, S3, S5,
×
1–
S7, S9, S11, S13, S15, S17, S19, S21, S23, S25, S27, S29, S31, S33 and S35.
MS analysis. Performed on MS Bruker microOTOF-QIII (Bruker Corporation, Billerica, MA, USA).
MS spectra of 1–18 peptides are available in Supplementary Materials: Figures S2, S4, S6, S8, S10, S12,
S14, S16, S18, S20, S22, S24, S26, S28, S30, S32, S34 and S36.
Preparative HPLC. Performed on a CombiFlash, EZPrep, Teledyne ISCO (Lincoln, Nebraska,
USA) using a Supelco Discovery BIO Wide Pore C18 column (25 cm
×
21.2 mm, 10 mm; Sigma-Aldrich);
flow rate, 5 mL/min; detection wavelengths, 220 and 254 nm) with gradient ratio A (0.1% TFA in
MeCN) and B (0.1% TFA in H2O) 0:100 to 18:82 in 30 min, followed by an isocratic run for 5 min.
Spectroscopic measurements. Performed on UV spectrophotometer Hitachi (Hitachi, Tokyo,
Japan), in a wavelength range from 400 nm to 800 nm. UV spectra of
Supplementary Materials: Figures S37–S54.
1–18 peptides are presented in
Fluorescence measurements. Performed on FLUOROMAX-3 Horiba Scientific (Edison, NJ, USA)
in a wavelength range from 470 nm to 600 nm, excitation wavelength 440 nm. Fluorescence intensity
spectra of 1–18 peptides are presented in Supplementary Materials: Figures S56–S73.
3.3. Spectroscopic Measurements with Congo Red (GP 5)
To initiate the aggregation process, peptide samples were incubated for 7 days at 37.4 ◦C in 1 mL of
phosphate buffer solution (concentration 0.1 M, pH 7.2), when difficulties in the solubility of peptides
in the buffer were observed, samples were sonicated for 15 s. The final concentration of the incubated
peptides was c = 1.44 mM. Subsequently, 1 mL of Congo Red (Sigma-Aldrich) solution (c = 45 µM,
phosphate buffer, pH 7.2) was added to samples, which were incubated for a further 4 days at room
temperature. During this period spectroscopic measurements were performed in the wavelength range
of 800 nm to 400 nm. Aggregation studies of all the incubated samples spectroscopic measurements
were begun 30 min after the addition of the Congo Red solution. Registered spectra of a mixture
containing 1 mL solution of Congo Red (c = 45
µM, phosphate buffer, pH 7.2) and of a 1 mL of solution
of phosphate buffer (concentration 0.1 M, pH 7.2), also incubated for 4 days at room temperature,
were used as controls. All UV–Vis spectra peptides
Supplementary Materials.
1–18 incubated with Congo Red are presented in
3.4. Spectroscopic Measurements with Thioflavin T (GP 6)
◦
To initiate the aggregation process, peptide samples were incubated for 7 days at 37.4 C in
2 mL of solution of phosphate buffer (concentration 0.1 M, pH 6.0), when difficulties in the solubility
of peptides in the buffer were observed, samples were sonicated for 15 s. To the samples was then
added 2 mL of Thioflavin T solution (c = 57 mM, phosphate buffer, pH 6.0). The samples were
incubated for another 4 days at room temperature. The final concentration of the incubated peptides
was c = 0.139 mM. Starting 30 min after the addition of Thioflavin T solution, over the following
days fluorescence measurements were performed in the wavelength range from 470 nm to 600 nm
(excitation
λ = 440 nm). Registered spectra of the mixture containing 2 mL solution of Thioflavin T
(c = 57 mM, phosphate buffer, pH 6.0) and 2 mL of phosphate buffer solution (concentration 0.1 M,
pH 6.0), also incubated for 4 days at room temperature, were used as the controls. All fluorescence
spectra peptides 1–18 incubated with Thioflavin T are presented in Supplementary Materials.

Molecules 2019, 24, 1600
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3.5. Microscopic Measurements (GP 7)
A sample for microscopic analysis (peptide and Congo Red solution) was centrifuged at
12,000–14,000 rpm in a centrifuge tube to pellet the fibrils, then washed three times with water.
The fibrils were then suspended in a small amount of water and placed on a glass microscope slide.
The sample was air-dried and analyzed under non-polarized and polarized light using a Delta Optical
Genetic Pro microscope (Warsaw, Poland).
3.6. AFM Studies
Atomic force microscopy (AFM) measurements were conducted in tapping mode using an
NT-MDT Solver PRO microscope (NT-MDT Spectrum Instruments, Moscow, Russia). Droplets of the
peptide samples were placed on glass holders, then dried in air for 5 h. Imaging was performed in
air using microfabricated silicon cantilevers (model TESP, force constant about 40 N/m) (NT-MDT
Spectrum Instruments, Moscow, Russia). The imaging software was NanoScope Software Version 5.
3.7. CD Studies
CD studies were performed with a Jasco J-1500 spectrometer (ABL and E-JASCO Polska, Cracow,
Poland). Far-UV CD experiments were performed using a J-1500 CD spectrometer (Jasco). The samples
were prepared in a phosphate buffer solution, pH 7.2, concentration 0.1 mg/mL (1 mg of each analyzed
peptide was dissolved in 10 mL of buffer solution). All studies were carried out at ambient temperature.
The dissolved samples were loaded in a rectangular quartz cuvette (1 mm path length, Hellma).
Spectra were collected in the range of 190–270 nm. Other experimental settings were as follows:
Data pitch, 5 nm; scanning mode, continuous; scanning speed, 100 nm/min; bandwidth, 3 nm;
integration time, 1 s.
4. Conclusions
The research presented here revealed new regions in the structure of insulin that may be involved
in its aggregation. Its findings could enable the more rational design of inhibitors in the hormone
aggregation process, which is crucial not only from the point of view of developing new drugs for the
treatment of diabetes, but also of designing additives for stabilizing insulin preparations. As reference
points, two fragments A13–A19 H–LYQLENY–OH (16), B12–B17 H–VEALYL–OH (17), which are
known hot spots of insulin and a fragment B22–B27 H–RGFFYT–OH (18) suspected of amyloidogenic
properties were used.
It was found that the synthesized peptide
4
(A11–A21) covering peptide 16 was able to aggregate.
(A17–A21) showed different aggregation
However, it is surprising that peptides (A11–A16) and
5
6
susceptibility. Fragment A11–A16 can be classified as aggregable (based on a positive ThT test result
and microscopic examination with an ambiguous CR assay result). However, fragment A17–A21
(peptide
fibrous structures.
In the case of the N-terminal fragment of the A-chain, for which di-peptide A3–A4 was pointed
as a fragment influencing aggregation, we found that penta-peptides and comprising fragment
6) forms mainly amorphous structures in the aggregation process, instead of the expected
2
3
1–10 meet all the criteria aggregating peptides. Regarding the B-chain of insulin, for which the
B12–B17 fragment is a known hot spot and the peptide susceptible to aggregation is the B22–B27
fragment, varying aggregation ability was also observed in the B11–B20 region (peptides 10, 11 and
12). For deca-peptide 10 (B11–B20) and penta-peptide 11 (B11–B15), microscopic studies revealed the
formation of fibrous structures. In the case of peptide 12 (B16–B20), microscopic images showed the
presence of amorphous and fibrous structures. In microscopic studies of the C-terminal region of
the B chain of insulin (peptides 13
B21–B25. The CR and ThT assays gave clear results confirming its susceptibility to aggregation. In the
N-terminal region of the B chain of insulin for peptides (B1–B10) and (B1–B5), characteristic fibrous
, 14 and 15), sole fiber structures were seen in the case of fragment
7
8

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structures were observed in microscopic studies. However, all tests (microscopic examination, CR and
ThT test) were positive only in the case of peptide . This finding is consistent with the modeling
result indicating that fragment B1–B5 may affect insulin aggregation. For deca-peptide (B1–B10) and
penta-peptide (B6–B10), the results of three independent tests were ambiguous. CD studies showed
that mature fibrils are formed via -helix rich structures from random coil and immature aggregates.
This observation is similar to what has been observed for -amyloid. Slow and gentle changes were
8
7
9
α
β
visible in the spectra, from random coils to more β rich structures. These changes were most marked
in fragments from chain B: B6–B10, B11–B20 and B1–B5.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/8/1600/s1,
the HPLC of crude 1–18 peptides (Figures S1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35); MS spectra of
1
–
18 peptides (Figures S2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36); UV spectra of
1–18 peptides
(Figures S37–S54) and fluorescence intensity spectra of 1–18 peptides (Figures S56–S73).
Author Contributions: M.S., J.F., J.W. and A.C. participated in peptide synthesis, self-assembling and CD studies;
M.S. and Z.J.K. contributed in preparation of manuscript and discussion of results; L.P. and L.S. performed AFM
studies; S.W. participated in the discussion of results; B.K. coordinated the research and wrote the paper.
Funding: This work was supported by the National Science Centre, Poland, under project number
UMO-2015/19/B/ST8/02594
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–18 are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).