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The structure of poly-L-lysine in different solvents

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Understanding the factors that affect the conformational stability of the polypeptide main chain provides insight not only into the molecular basis of unfolded states but also into the earliest event that occurs during the protein folding. The presented study was concentrated on finding the conformational distributions of poly-l-lysine (PLL) by applying infrared spectroscopy. We assigned the amide bands for different conformations of PLL in water. At low pH values PLL mainly possesses the PII and β structures while at higher pH values and low temperatures characteristic bands for the α-helical conformation are found. The increase in temperature induces the formation of β structures. The obtained assignment of the infrared bands for various conformations was used to determine the conformational populations of PLL in non-aqueous solvents. In TFE, PLL possesses an α-helix structure that is after heating partially transformed into the PII conformation. DMSO enables a uniform α-helical conformation of PLL. A similar uniform conformation (PII, 88%) was found for PLL dissolved in ethylene glycol, suggesting that the PII structure is not limited to the presence of water molecules or charged side chains. The role of intermolecular interactions between the solvent molecules and PLL in stabilizing the PII conformation is discussed.
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The structure of poly-L-lysine in different solvents
Andreja Mirtič
a
,Jože Grdadolnik
a,b,
a
National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia
b
EN-FIST, Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia
HIGHLIGHTS
The structures of PLL was determined
by analysis of amid I, II and III regions.
High population of PII structure was
found in PLL dissolved in non-aqueous
medium.
P
II
is stabilized in ethylene glycol by H-
bonds between solvent and peptide.
TFE and DMSO stabilize α-helical
structure of PLL.
GRAPHICAL ABSTRACT
abstractarticle info
Article history:
Received 24 January 2013
Received in revised form 13 February 2013
Accepted 13 February 2013
Available online 22 February 2013
Keywords:
Poly-L-lysine
PII
Alpha-helix
Beta-conformation
ATR infrared spectroscopy
DFT calculation
Water
DMSO
TFE
Ethylene glycol
Understanding the factors that affect the conformational stability of the polypeptide main chain provides
insight not only into the molecular basis of unfolded states but also into the earliest event that occurs during
the protein folding. The presented study was concentrated on nding the conformational distributions of
poly-L-lysine (PLL) by applying infrared spectroscopy. We assigned the amide bands for different conforma-
tions of PLL in water. At low pH values PLL mainly possesses the P
II
and βstructures while at higher pH values
and low temperatures characteristic bands for the α-helical conformation are found. The increase in temper-
ature induces the formation of βstructures. The obtained assignment of the infrared bands for various
conformations was used to determine the conformational populations of PLL in non-aqueous solvents. In
TFE, PLL possesses an α-helix structure that is after heating partially transformed into the P
II
conformation.
DMSO enables a uniform α-helical conformation of PLL. A similar uniform conformation (P
II
, 88%) was
found for PLL dissolved in ethylene glycol, suggesting that the P
II
structure is not limited to the presence of
water molecules or charged side chains. The role of intermolecular interactions between the solvent mole-
cules and PLL in stabilizing the P
II
conformation is discussed.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Understanding the mechanism of peptide folding at the molecular
level is a subject currently under intensive study. A dynamic peptide
Biophysical Chemistry 175-176 (2013) 4753
Correspon ding author at: National Institute of Chemi stry, Hajdrihova 19, SI-1000
Ljubljana, Slovenia.
E-mail address: joze.grdadolnik@ki.si (J. Grdadolnik).
0301-4622/$ see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bpc.2013.02.004
Contents lists available at SciVerse ScienceDirect
Biophysical Chemistry
journal homepage: http://www.elsevier.com/locate/biophyschem
Author's personal copy
structure with a large population of unstructured regions requires
spectroscopic methods that provide detailed structural information
probed at a very short timescale (picoseconds). Infrared spectroscopy
is a powerful method in structural biology capable of monitoring struc-
tures with such short life times. However, to determine the various
types of particular secondary structural elements, a uniform and de-
tailed assignment of the spectral bands needs to be resolved rst. We
used FTIR spectroscopy to probe the distribution of various conforma-
tions of the homopolypeptide poly-L-lysine (PLL). PLL has positively
charged side chain amino groups at a neutral and low pH resulting in
electrostatic repulsion between them that allows only the extended
conformation of polypeptides. The secondary structure of PLL has
been characterized by numerous spectroscopic methods such as CD
[1,2],IR[3],NMR[46], Raman optical activity [79],VCD[10] and
most recently by UV resonance Raman spectroscopy [1113].Byusing
UV resonance Raman spectroscopy (UVRS), Mikhonin et al. [14] dem-
onstrated the conformation of PLL at pH 2 as an equilibrium between
P
II
and the extended β-strand conformations in the ratio 60:40, respec-
tively. The P
II
conformation, described by Tiffany and Krimm [1],is
characterized by the lack of intramolecular hydrogen bonds. The factors
that stabilize P
II
conformation are not well established [15]. It was pro-
posed that P
II
-helical conformation arises from the combination of min-
imization of unfavorable intrachain steric interactions and favorable
backbone solvation [16,17], even though the role of water remains de-
bated. The resulting opened and rather exible structure is becoming
increasingly recognized as general structural characteristics of natively
unfolded proteins [18,19] and as a candidate for the intermediate struc-
ture in amyloid bril formation [13,20]. Moreover, it has been shown
that the P
II
structure is one of the most preferential conformations
found in dipeptides in water [21,22].
In the present article, we not only determine the distributionsof the
conformations of PLL in water but also in several other solvents. The
applied solvents preferentially stabilize one conformation by changing
the preference of inter- and intramolecular hydrogen bond formation.
Determination of the structure by the application of infrared spectros-
copy is mainly based on analysis of the amide I region. This region in
general contains bands that are strongly overlapped, especially in the
case when the polypeptide establishes several distinct conformations.
To ensure the reliable determination of the peptide (protein) structure,
additional indicative bands sensitive toparticular structures are needed.
Thus, we introduced new structural parameters retrieved from the in-
frared spectra, which are able to distinguish between the α-helix and
P
II
conformations, as well as between the various types of βstructures,
such as βconformation, β-strands and β-sheets, by analyzing the amide I
and amide II regions of peptide infrared spectra, as well as amide III.
2. Materials and methods
Poly-L-lysine (PLL) HCl was purchased from Sigma (MW
MALLS
=
31185 Da) and used without further purication. The samples were
freshly prepared in H
2
OorD
2
O (Cambridge Isotope Laboratories,
Inc.) as a 4 wt.% solution. The pH was adjusted to pH 4 and pH 11.6
using appropriate amounts of HCl (DCl) and NaOH (NaOD). The solu-
tions in DMSO (Sigma-Aldrich), DMSO-d6 (Cambridge Isotope Labora-
tories, Inc.), TFE (Sigma-Aldrich) and ethylene glycol (Sigma-Aldrich)
were prepared as 4 wt.% concentrations.
2.1. Fourier transform infrared spectroscopy
The infrared spectra were measured using the PerkinElmer System
2000 and the Bruker Vertex 80 infrared spectrometers. Spectra were
recorded in the range between 7000 cm
1
and 450 cm
1
in ATR
mode. The spectra obtained from the ATR-infrared measurements
were rst recalculated to get the pure absorption spectra [23]. Before
any spectral analysis, the spectrum of pure water was subtracted. The
band overlapped regions were analyzed using the Grams band tting
procedure by optimizing the sum of the bands with the mixed
Lorentzian and Gaussian shapes. The solution spectra of PLL were mea-
sured in a temperature range from 10 °C to 80 °C using a diamond ATR
cell (Specac) equipped with a heated top plate. To reduce the strong
bands due to the absorption of diamond, backgrounds were collected
for each recorded temperature. The assignment of the structural sensi-
tive bands in amide I, II, and III regions is based on previous results on
blocked dipeptides [22] and vibrational spectra of proteins in solution
with known structure [2428].
2.2. Ab initio calculations of the pKa
The ab initio pKa calculations of propylamine in the different solvents
were performed for the gas phase and in solution using Gaussian 09 [29].
The solvation energies have been calculated using the Polarisable Contin-
uum Model (PCM) [30] at the B3LYP/6-31++G(d,p) [3133] level.
3. Results and discussion
3.1. The assignment of the major spectral features in the FTIR spectra of
the different conformers of PLL
The conformational sensitive regions in the infrared spectrum of
polypeptides are known as amide I (1600 cm
1
1690 cm
1
), amide
II (1500 cm
1
1580 cm
1
) and amide III (1220 cm
1
1330 cm
1
).
The amide I mode has a predominant contribution of backbone C_O
stretching. Its frequency is sensitive to the conformation of the peptide
backbone, particularly to various types of secondary structures. Since
the backbone C_O group acts as a proton acceptor, the frequency of
the amide I band possesses a great sensitivity to variations in the envi-
ronment of the C_O groups. The establishing of hydrogen bonds and
the extension of inter-residual vibrational coupling [12,34] may also in-
uence the frequency and shape of amide I vibration. The amide II mode
is less sensitive to the various secondary structural elements. However,
its frequency reects the participation of backbone NH groups in the
various types of hydrogen bonds with proton acceptor groups from
the protein or molecules from the protein environment. The amide III
region is the most sensitive indicator for the peptide conformations.
The bands from the amide III region, which chieyresultfromN\H
in-plane bending mixed with C\CandC\N stretching, C_O in-plane
bending and C
α
\H bending, possess information on the values of the
ϕand Ψtorsion angles from the peptide backbone [22].
3.2. PLL in water (pH 11.6)
A high pH value (11.6) and low temperature stabilize the α-helical
conformation of PLL in water. The band tting algorithm resolves the
amide I band on two band components at 1664 cm
1
and 1644 cm
1
respectively (Fig. 1). The strongest model band at lower wavenumbers
is ascribed to the α-helix conformation. The band frequency of this
amide I band is located close to the α-helix band found in the infrared
spectrum published by Dzwolak et al. [26]. However, in the α-helical
state, there is an additional band in the amide I region at 1664 cm
1
,
the assignment of which is not conclusive. One of the possibilities,
which should be proved by the presence of corresponding bands in
the amide II and amide III regions, is that this amide I band belongs to
turns [35]. The decomposition of the amide II bandrevealed two prevail-
ing components located at 1550 cm
1
and 1572 cm
1
.Thesemodel
bands are assigned to the α-helix conformation and the amide II coun-
terpart of the 1664 cm
1
amide I band (Fig. 1). The remaining bands
presented in Fig. 1 are due to the NH
2
symmetric and antisymmetric
deformation of the lysine side chain located at 1602 cm
1
and
1532 cm
1
respectively. The remaining band in the amide I region lo-
cated at 1628 cm
1
is attributed to the bending vibration of water mol-
ecules, which remains after the subtraction of the bulk water.
48 A. Mirtič, J. Grdadolnik / Biophysical Chemistry 175-176 (2013) 4753
Author's personal copy
The band structure of the extended amide III region is presented in
Fig. 2. At the highfrequency region, two sharp model bands are assigned
to the CH
2
(1394 cm
1
)andC
α
\H (1348 cm
1
) bending vibrations.
The present assignment of the band at 1348 cm
1
is not unique.
While Barron et al. [8] assigned this band as a component of the
amide III region representing the vibration of hydrated α-helices, vibra-
tional studies of short dipeptides showed that this band isalready out of
the amide III region [22]. It is predominately attributed to C
α
Hbending
mixed with the amide III mode but has no potentials for the structural
characterizations of peptides [36]. Therefore, the amide III region of
the PLL is composed of two band components. The strongest band at
1295 cm
1
has been assigned to the α-helical conformation of the
polypeptide. The band near 1300 cm
1
is generally found in the vibra-
tional spectra of globular proteins composed mainly of α-helices
[3739]. Moreover, the component of the amide III vibration in the in-
frared spectra of dipeptides, which was assigned to an αconformation
of the peptide backbone [22], wasfound at a similar position.The inten-
sity of the second band in the amide III region located at 1257 cm
1
(Fig. 2) is much lower compared to the central band at 1295 cm
1
.Its
appearance is correlated to the bands at 1664 cm
1
and 1572 cm
1
in the amide I and amide II regions respectively, which were assigned
to turn structures.
The area of the component bands can be efciently applied for a
rough estimation of the populations of a particular conformation.
This analysis revealed that the population of α-helix in PLL at 10 °C
and at pH 11.6 is between 78% and 81% as obtained from the amide
I or amide III region, respectively. These results are in accordance
with results published by Jiji et al. [13].
At higher temperatures and pH 11.6, helical PLL gradually transforms
into the β-sheet conformation. The infrared spectrum of β-sheet PLL
(Fig. 3) shows two characteristic amide I bands, weaker at 1692 cm
1
and stronger at 1618 cm
1
that are typical for the (aggregated) antipar-
allel β-sheet (Table 1).
In the decomposed amide III region presented in Fig. 4, three compo-
nents of different βconformations can be observed; the rst component
is located at 1270 cm
1
, which is assigned solely to amino acidsin the β
conformation that are not involved in the secondary structure [22],the
second is located at 1243 cm
1
and is assigned to the β-strands and the
third is located at 1222 cm
1
and is assigned to the strong hydrogen
bonded β-sheets of aggregated PLL. The frequency of the second com-
ponent is close to the value of the amide III band found in the infrared
spectra of βstructured proteins [7], while the amide III components
near 1220 cm
1
have already been found in the spectra of aggregated
proteins [7,25,40,41]. The bands representing the β-sheet conformation
populates 61% and 58% of all the conformations obtained from the
amide I and III regions respectively. The structure of the amide I,
amide II and amide III bands revealed that besides the prevailing
β-sheets, the nal state of the brillation of PLL also contains some
amino acids in the turn, α-helical and P
II
conformations.
3.3. PLL in water (pH 4)
The poly-L-lysine side chains at pH 4 are fully ionized, so electro-
static repulsion prevents the formation of the α-helical conformation.
The characteristic infrared spectrum of PLL in a water solution at
10 °C and a low pH shows broadened and asymmetrical amide I and
amide II bands (Fig. 5). Similar to that at a higher pH, the tting algo-
rithm is used to determine the intrinsic components of the amide
bands as shown in Fig. 5. The band analysis shows that the major
Fig. 1. The infraredspectrum of PLL at pH 11.6 and T= 10 °C with tted model bandsthat
represent: the α-helix (1644 cm
1
,1550cm
1
), the turn conformation (1664 cm
1
,
1572 cm
1
), the CH
2
and Cα\H vibrations (1508 cm
1
,1489cm
1
,1474cm
1
,
1461 cm
1
,1443cm
1
), the NH
2
groups of the lysine side chain (1602 cm
1
,
1532 cm
1
), and the water bending (1628 cm
1
).
Fig. 2. The infrared spectrum in an amide III region of PLL at pH 11.6 and T =10 °C with
tted model bands that represent: the α-helix (1295 cm
1
), the turn conformation
(1257 cm
1
), and the CH
2
and C
α
\H vibrations.
Fig. 3. The infraredspectrum of PLL at pH 11.6 and T= 80 °C with tted model bandsthat
represent: the β-sheet (1692 cm
1
,1618cm
1
,1530cm
1
), the turn conformation
(1669 cm
1
, 1563 cm
1
), the P
II
conformation (1652 cm
1
,1543cm
1
), the α-helix
(1642 cm
1
,1553cm
1
), CH
2
and Cα\H vibrations (1495 cm
1
,1477cm
1
,
1458 cm
1
,1444cm
1
), the NH
2
groups of the lysine side chain (1600 cm
1
,
1508 cm
1
), the water bending (1629 cm
1
), and the CH
2
and C
α
\Hvibrations.
Table 1
The assignment of the model bands from the amide I, II and III region, retrieved with
the band tting algorithm from the corresponding spectral regions of PLL in water.
Conformation Frequency (cm
1
)
Amide I Amide II Amide III
Turn 16641674 15631572 12571260
P
II
16481654 15431546 13081311
a
α16431645 15501552 12901295
a
β-Conformation / / 12701280
a
β-Strand 16251630
b
/ 12401243
β-Sheet 16301640
c
/ 1230
Aggregated β-sheets 1692, 1618 1530 12191222
a
Obtained from Grdadolnik et al. [22].
b
Obtained from Huang et al. [27].
c
Obtained from Baumruk et al. [28].
49A. Mirtič, J. Grdadolnik / Biophysical Chemistry 175-176 (2013) 4753
Author's personal copy
population of PLL at pH 4 is the P
II
conformation (45%) followed by
the β-structures. The amide I and II band components attributed to
P
II
are located at 1649 cm
1
and 1547 cm
1
respectively. The corre-
sponding bands for βstructures can be found at 1618 cm
1
and
1522 cm
1
, while small bands at 1644 cm
1
and 1671 cm
1
were
assigned to the α-helix and turn structures. The amide II counterpart
of the later structure is located at 1570 cm
1
. The bands at 1594 cm
1
and 1506 cm
1
are assigned to the NH
3
+
stretching of the lysine side
chains. The assignment of those bands was checked with deuterium
substitution of the solvent (data not shown).
Similar structural elements of PLL conformation in water at a low
pH was found by decomposing the amide III region. The extended amide
III region of the FTIR spectrum of PLL in water at pH 4 shows at least four
well-dened bands with the frequencies 1311 cm
1
(P
II
), 1291 cm
1
(α-helix), 1272 cm
1
(βconformation), 1257 cm
1
(turn), 1243 cm
1
(extended β-strand) and 1219 cm
1
(aggregated β-sheet). However, it
needs to be emphasized that the decomposition of the amide III region
presented in Fig. 6 yielded slightly different distributions of the
presented conformers. By comparison of the integral intensity, the
ratio of the P
II
conformation falls to 25%. The reason for this inconsisten-
cy is not known. The comparison of the results obtained from the anal-
ysis of amide I and amide III regions reveals that the decomposition of
the latter region retrieves more complete information about the
populations of different conformers of PLL. The band components of
the amide III region are more resolved already in the original spectrum.
Consequently, the application of the band tting algorithm is therefore
more reliable. This is the reason why we are able to determine some
conformations connected to the β-structure, in which the characteristic
patterns are strongly overlapped in the amide I region with water and
NH
3
+
bending.
The temperature dependence of the PLL conformation at pH 4 is
not as extensive as at pH 11.6. It culminates in a small upshift of the
amide I band that corresponds to a small downshift of the amide II
band. The frequency of the amide II band is the most sensitive for
the formation of hydrogen bonds and consequently to hydration
[42]. Therefore the change in hydration of PLL causes a downshift of
this band by 16 cm
1
. This temperature dependence derives from the
hydrogen bonds of water molecules to amide carbonyl andN\H groups
[4244]. With a higher temperature, the wateramide hydrogen bond
strengths decrease, which consequently form stronger carbonyl bond
resonance and weaker C(O)\N bonding resonance of the peptide
bond. Comparison of the band intensities of the bands corresponding
to the P
II
conformation reveals that heating PLL at a low pH from
20 °C to 80 °C decreases the population of P
II
by roughly 10%.
3.4. pKa calculation in different solvents
The experimental values of pKa for PLL in TFE and ethylene glycol
are not known. The protonation state of the side chain amino group of
PLL in different solvents was determined indirectly by comparison
with the calculated pKa values of model molecule propylamine
using the DFT approach. Propylamine was used as a model molecule
for the side chain of lysine possessing a similar pKa value in water
as PLL. Solvation free energy was calculated using the polarized con-
tinuum model (PCM) with the B3LYP/6-31++G(d,p) method and
calculated using the equation [45]:
ΔΔG¼ΔGGP PropylNH3
þ

ΔGGP PropylNH2
ðÞþΔGsolvent PropylNH3
þ

ΔGsolvent PropylNH2
ðÞþΔGGP solventHþ

ΔGGP solventðÞ
þΔGsolvent solventHþ

ΔGsolvent solventðÞ
ð1Þ
The obtained ΔΔG values were then used to calculate the pKa
values, which were compared to experimentally determined pKa
values for propylamine in different solvents (Table 2).
A very good agreement with the experimental values was obtained
for water and DMSO, suggesting a rather good match between the
calculated pKa value for ethylene glycol and TFE. The low values of the
calculated pKa of propylamine in ethylene glycol and TFE suggest the
deprotonation of amino groups from side chains in both solvents.
Fig. 4. The infrared spectrum in an amide III region of PLL at pH 11.6 and T =80 °C with
tted model bands that represent: βconformations (βconformation at 1270 cm
1
,
β-strand at 1243 cm
1
, hydrogen bonded β-sheet at 1222 cm
1
), the turn conforma-
tion (1258 cm
1
), the P
II
conformation (1308 cm
1
), the α-helix (1290 cm
1
), and
the CH
2
and C
α
\H vibrations.
Fig. 5. The infrared spectrum of PLL at pH 4 and T =25 °C with tted model bands that
represent: the β-sheet (1618 cm
1
, 1522 cm
1
), the turn conformation (1671 cm
1
,
1570 cm
1
), the P
II
conformation (1649 cm
1
, 1547 cm
1
), the α-helix (1644 cm
1
),
the NH
3
+
groups of lysine side chain (1594 cm
1
, 1506 cm
1
), the water bending band
(1630 cm
1
), and the CH
2
and C
α
\H vibrations.
Fig. 6. The amide III region of the infrared spectrum of PLL, pH 4, at 20 °C tted with model
bands that represent: βconformations (βconformations at 1272 cm
1
,β-strands at
1243 cm
1
, aggregated β-sheet at 1219 cm
1
),theturnconformation(1257cm
1
), the
P
II
conformation (1311 cm
1
), the α-helix (1291 cm
1
), and the CH
2
and C
α
\Hvibrations.
50 A. Mirtič, J. Grdadolnik / Biophysical Chemistry 175-176 (2013) 4753
Author's personal copy
3.5. PLL in different solvents
To conrm the presented assignment and to elucidate the origin of
the bands of individual conformation, we extended the conformational
studies to cover various solvents that are able to promote particular
types of conformations. We used the common α-helix promoting sol-
vent TFE to stabilize the α-helix conformation of PLL [48,49].PLLwasti-
trated with different ratios of water:TFE at pH 4. With an increasing TFE
concentration from 0% to 80% (v:v), the spectra of PLL remain unaffect-
ed. An additional increase of the TFE concentration induces a shift of the
amide I 3band maximum from 1649 cm
1
to 1652 cm
1
, indicative of
P
II
/α-helix transformation, i.e. the mainly P
II
conformation of PLL in
water converts to the α-helical conformation in 100% TFE. The calculat-
ed amount of α-helix conformation in 100% TFE is 84%. It should be
mentioned that the amide I band characterizing an α-helix of PLL is
blue-shifted in TFE due to the low dielectric constant of TFE compared
to water. Heating PLL in 100% TFE from 10 °C to 70 °C results in a de-
crease of the α-helix conformation by 13%, which runs parallel with
the formation of the P
II
conformation at 1659 cm
1
(Fig. 7). These re-
sults are consistent with the data published by Xiong and Asher [50],
where polyalanine peptide in 50% TFE, which possesses mainly helical
conformation, melts 9.1% of its α-helices into P
II
after heating the pep-
tide from 25 to 40 °C.
We measuredPLL in DMSO as a solvent with only hydrogen bond ac-
ceptor abilities. At low temperatures, PLL in DMSO exhibits an even
higher α-helical population than in TFE, i.e. a solvent with hydrogen
bond donor abilities. The spectrum of PLL in DMSO is characterized by
a sharp amide I band at 1650 cm
1
, an amide II band at 1548 cm
1
(Fig. 8) and an amide III band at 1295 cm
1
. The amide III region is
only applicable when the DMSO solvent is substituted with DMSO-d
6
.
The amide I band is upshifted due to the low dielectric constant of
DMSO. Contrary to the data that DMSO disrupts or weakens the intra-
molecularhydrogen bonds [51], ourresults indicate that DMSO strongly
stabilizes the α-helix conformation, reaching 97% of the population in
PLL. It was shown that polar amino acid side chains have dipoledipole
interactions with the oxygen atom of DMSO and form hydrogen bonds,
whereas apolar side chain alkyl groups become solvated by DMSO
through the formation of a hydrophobic pocket [52]. The dual solvation
properties of DMSO cause it to be a good membrane-mimicking solvent
[52].
Ethylene glycol was the next solvent where the structure of PLL was
probed. As a solvent it has many applications especially as a cryogenic liq-
uid. Calculation of the pKa values showed that the ε-amino group of the
lysine side chain in ethylene glycol has a signicant potential to be
deprotonated. The reduced electrostatic repulsions of the side chains of
PLL in ethylene glycol enable the stabilization of P
II
conformation com-
pared to water, where repulsions between ionized ε-amino groups pro-
motes extended β-strand conformation [14]. PLL in ethylene glycol
populates 88% of the P
II
conformation, as can be seen in Fig. 9.The
bands characteristic of the P
II
conformation are located at 1656 cm
1
in
the amide I region, 1546 cm
1
in the amide II region, and 1311 cm
1
in the amide III region. Due to the low dielectric constant of ethylene gly-
col, the amide I band characterizing the P
II
conformation of PLL is
blue-shifted compared to the amide I position in water. From the amide
III region in Fig. 9, a band is observed at 1238 cm
1
that corresponds to
the β-strand structure. After heating PLL in ethylene glycol from 10 to
65 °C, 10% of the P
II
conformation melts as observed from the difference
spectra.
3.6. Stabilization of the P
II
conformation
Vibrational studies of the Lys dipeptide showed that the amino acid
lysine possesses a strong propensity to P
II
conformation. A recent study
revealed that Lys in a short dipeptide populates 55% of P
II
conformation
[22]. This high propensity of lysine to adopt the P
II
conformation was
explained by the strong backbone electrostatic interactions and by the
screening of those interactions with water molecules, which is
inuenced by the side chain [21,22].TheP
II
conformation is supposed
to be stabilized by water hydrogen bonding to the backbone amide
and carbonyl group [53]. However, Drozdov et al. found no evidence
for a role of water bridges in stabilizing P
II
[17]. Contrary, Nerenberg
and Head-Gordon found that the P
II
conformation is optimizing the
packing of water molecules in the hydration shell of the peptide [54].
Table 2
pKa values for the propylamine model molecule in different solvents obtained ab initio
from Eq. (1) and experimental values.
Solvent pKa calculated pKa experimental
Water 10.56 10.6
a
TFE 4.54 /
DMSO 9.76 10.4
b
Ethylene glycol 1.82 /
a
Obtained from Perrin et al. [46].
b
Obtained from Makowska et al. [47].
Fig. 7. The melting of the α-helix and the formation of the P
II
-helix with increasing
temperature relative to the α-helix and P
II
content of PLL in TFE at 20 °C.
Fig. 8. Spectra of PLL in 100% TFE (black curve) and in DMSO (gray curve) at 20 °C.
Fig. 9. Spectra of PLL in ethylene glycol (black curve) and in water at pH 4 (gray curve).
51A. Mirtič, J. Grdadolnik / Biophysical Chemistry 175-176 (2013) 4753
Author's personal copy
The water molecules reduce the attractive electrostatic interactions be-
tween the peptide atoms, leaving the steric interactions as the main
force for determining conformational preferences [15].P
II
minimizes
these interactions and thus becomes the most prominent conformation.
However, in this study the P
II
conformation was determined in a
non-aqueous solvent for the rst time. Moreover, Law and Daggett
[55] found no correlation between the dielectric constant of the solvent
and the P
II
structure. Thus we suggest that the stabilization of the P
II
struc-
ture in ethylene glycol occurs due to the deprotonated side chain of the
lysine leading to a more sterically optimized structure of the P
II
-helix.
The question of Why there is no formation of the P
II
-helix in TFE and
DMSO, where PLL also has a deprotonated ε-aminedenitely needs an
answer. Ethylene glycol has, like a water molecule, both proton donor
and proton acceptor groups that are able to form intermolecular hydro-
gen bonds with the NH and CO group of the peptide backbone and thus
hinder the formation of the intramolecular hydrogen bonds characteristic
of α-helices.
4. Conclusions
We used infrared spectroscopy to characterize the spectral bands
of individual conformational states of PLL stabilized by different
solvents. PLL changes its conformation depending on the solvent, pH
value and temperature. In water at a low pH, it is populated predomi-
nately by the P
II
conformation. The increase of the pH value stabilizes
the α-helical structure, which transforms into the aggregated β-sheet
structure after heating the peptide (Fig. 10).
The conformation of PLL is radically changed by the addition of
TFE, ethylene glycol or DMSO. The analysis of the amide I, II and III
bands revealed that TFE and DMSO induced the α-helix conforma-
tion, while ethylene glycol, like water, induced the P
II
conformation.
Thus, the P
II
conformation is not connected only to water as a solvent
and the hydration of peptide bonds. Ethylene glycol even more ef-
ciently stabilizes the P
II
-helix. We suppose that this stabilization has
similar roots as predicted for water, i.e. the main forces that stabilize
the P
II
conformation are steric interactions of the uncharged side
chains of PLL and the establishment of hydrogen bonds between the
CO and NH groups from the peptide backbone and the solvent's pro-
ton donor and acceptor groups.
The conformational analysis of PLL in various solvents showed that
the decomposition of the amide III region provides a more detailed dis-
tribution of the peptide conformations. Less extensive overlapping of
the characteristic bands that belong to a particular conformation im-
prove the accuracy of the structural determination. This is especially
true for two typical helical secondary structural elements, i.e. P
II
and
α. Both conformations have well separated bands in the amide III re-
gion, the frequencies of which are less sensitive to the type of solvent
compared to the corresponding amide I bands.
Acknowledgements
This work was supported by a grant from the Ministry of Educa-
tion, Science, Culture and Sport of the Republic of Slovenia. We
thank Franc Avbelj and Rok Borštnar for stimulating discussions.
References
[1] M.L. Tiffany, S. Krimm, New chain conformations of poly(glutamic acid) and poly-
lysine, Biopolymers 6 (1968) 13791382.
[2] J.J. Grigsby, H.W. Blanch, J.M. Prausnitz, Effect of secondary structure on the
potential of mean force for poly-L-lysine in the alpha-helix and beta-sheet confor-
mations, Biophysical Chemistry 99 (2002) 107116.
[3] P.C. Painter, J.L. Koenig, Solution conformation of poly(L-lysine) Raman and
infrared spectroscopic study, Biopolymers 15 (1976) 229240.
[4] A. Darke, E.G. Finer, Nmr-studies of mixtures of poly-L-lysine hydrobromide with
water, Biopolymers 14 (1975) 441455.
[5] F.J. Joubert, N. Lotan, H.A. Scheraga, A nuclear magnetic resonance study of the
helix coil transition of poly L lysine in methanol water solvents, Physiological
Chemistry and Physics 1 (1969) 348.
[6] B. Perly, Y. Chevalier, C. Chachaty, Nmr and electron-spin-resonance study of the
conformations and dynamical properties of poly(L-lysine) in aqueous-solutions,
Macromolecules 14 (1981) 969975.
[7] I.H. McColl, E.W. Blanch, A.C. Gill, A.G. Rhie, M.A. Ritchie, L. Hecht, K. Nielsen, L.D.
Barron, A new perspective on beta-sheet structures using vibrational Raman optical
activity: from poly(L-lysine) to the prion protein, Journal of the American Chemical
Society 125 (2003) 1001910026.
[8] L.D. Barron, L. Hecht, E.W. Blanch, A.F. Bell, Solution structure and dynamics of
biomolecules from Raman optical activity, Progress in Biophysics and Molecular
Biology 73 (2000) 149.
[9] G. Wilson, L. Hecht, L.D. Barron, Vibrational Raman optical activity of alpha-helical
and unordered poly(L-lysine), Journal of the Chemical Society, Faraday Transactions
92 (1996) 15031509.
[10] T.A. Keiderling, R.A.G.D. Silva, G. Yoder, R.K. Dukor, Vibrational circular dichroism
spectroscopy of selected oligopeptide conformations, Bioorganic & Medicinal
Chemistry 7 (1999) 133141.
[11] S. Song, S.A. Asher, UV resonance Raman studies of peptide conformation in
poly(L-lysine), poly(L-glutamic acid), and model complexes: the basis for protein
secondary structure determinations, Journal of the American Chemical Society
111 (1989) 42954305.
[12] Y. Wang, R. Purrello, S. Georgiou, T.G. Spiro, UVRR spectroscopy of the peptide
bond. 2. Carbonyl H-bond effects on the ground- and excited-state structures
of N-methylacetamide, Journal of the American Chemical Society 113 (1991)
63686377.
[13] R.D. JiJi, G. Balakrishnan, Y. Hu, T.G. Spiro, Intermediacy of poly(L-proline) II
and β-strand conformations in poly(L-lysine) β-sheet formation probed by
temperature-jump/UV resonance Raman spectroscopy, Biochemistry 45 (2005)
3441.
[14] A.V. Mikhonin, N.S. Myshakina, S.V. Bykov, S.A. Asher, UV resonance Raman
determination of polyproline II, extended 2.5(1)-helix, and beta-sheet Psi angle
energy landscape in poly-L-lysine and poly-L-glutamic acid, Journal of the American
Chemical Society 127 (2005) 77127720.
[15] S. Toal, O. Amidi, R. Schweitzer-Stenner, Conformational changes of trialanine
induced by direct interactions between alanine residues and alcohols in binary
mixtures of water with glycerol and ethanol, Journal of the American Chemical
Society 133 (2011) 1272812739.
[16] R.V. Pappu, G.D. Rose, A simple model for polyproline II structure in unfolded
states of alanine-based peptides, Protein Science 11 (2002) 24372455.
[17] A.N. Drozdov, A. Grosseld, R.V. Pappu, Role of solvent in determining conforma-
tional preferences of alanine dipeptide in water, Journal of the American Chemical
Society 126 (2004) 25742581.
[18] L.D. Barron, E.W. Blanch, L. Hecht, Unfolded proteins studied by Raman optical
activity, Unfolded Proteins 62 (2002) 5190.
[19] Z.S. Shi, R.W. Woody, N.R. Kallenbach, Is polyproline II a major backbone confor-
mation in unfolded proteins? Unfolded Proteins 62 (2002) 163240.
[20] E.W. Blanch, L.A. Morozova-Roche, D.A. Cochran, A.J. Doig, L. Hecht, L.D. Barron, Is
polyproline II helix the killer conformation? A Raman optical activity study of the
amyloidogenic prebrillar intermediate of human lysozyme, Journal of Molecular
Biology 301 (2000) 553563.
[21] F. Avbelj, S.G.Grdadolnik, J. Grdadolnik, R.L. Baldwin, Intrinsic backbone preferences
are fully present in blocked amino acids, Proceedings of the National Academy of
Sciences of the United States of America 103 (2006) 12721277.
[22] J. Grdadolnik, V. Mohacek-Grosev, R.L. Baldwin, F. Avbelj, Populations of the three
major backbone conformations in 19 amino acid dipeptides, Proceedings of
the National Academy of Sciences of the United States of America 108 (2011)
17941798.
[23] J. Grdadolnik, ATR-FTIR spectroscopy: its advantages and limitations, Acta Chimica
Slovenica 49 (2002) 631642.
[24] S. Peternel, J. Grdadolnik, V. Gaberc-Porekar, R. Komel, Engineering inclusion
bodies for non denaturing extraction of functional proteins, Microbial Cell Factories
7 (2008) 34.
[25] S. Jevsevar, V. Gaberc-Porekar, I. Fonda, B. Podobnik, J. Grdadolnik, V. Menart,
Production of nonclassical inclusion bodies from which correctly folded protein
can be extracted, Biotechnology Progress 21 (2005) 632639.
Fig. 10. Schematic representation of conformational changes of PLL induced by changing
a pH value and temperature.
52 A. Mirtič, J. Grdadolnik / Biophysical Chemistry 175-176 (2013) 4753
Author's personal copy
[26] W. Dzwolak, V. Smirnovas, A conformational α-helix to β-sheet transition accom-
panies racemic self-assembly of polylysine: an FT-IR spectroscopic study,
Biophysical Chemistry 115 (2005) 4954.
[27] R. Huang, V. Setnicka, M.A. Etienne, J. Kim, J. Kubelka, R.P. Hammer, T.A.
Keiderling, Cross-strand coupling of a β-hairpin peptide stabilized with an
Aib-Gly turn studied using isotope-edited IR spectroscopy, Journal of the American
Chemical Society 129 (2007) 1359213603.
[28] V. Baumruk, P. Pancoska, T.A. Keiderling, Predictions of secondary structure using
statistical analyses of electronic and vibrational circular dichroism and Fourier
transform infrared spectra of proteins in H
2
O, Journal of Molecular Biology 259
(1996) 774791.
[29] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li,
H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, T. Vreven, J.A. Montgomery, J.E. Peralta, F. Ogliaro, M. Bearpark,
J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K.
Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega,
J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W.
Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J.
Dannenberg, S. Dapprich, A.D. Daniels, Farkas, J.B. Foresman, J.V. Ortiz, J.
Cioslowski, D.J. Fox Gaussian 09, Revision B.01 in Wallingford CT.(2009).
[30] J.B. Foresman, E. Frisch, Exploring Chemistry with Electronic Structure Methods,
Gaussian, Pittsburgh, Pa, 1996.
[31] C. Lee, W. Yang, R.G. Parr, Development of the ColleSalvetti correlation-energy
formula into a functional of the electron density, Physical Review B 37 (1988)
785789.
[32] A.D. Becke, Density-functional exchange-energy approximation with correct
asymptotic behavior, Physical Review A 38 (1988) 30983100.
[33] A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange,
Journal of Chemical Physics 98 (1993) 56485652.
[34] J.C. Austin, T. Jordan, T.G. Spiro, Ultraviolet resonance Raman studies of proteins
and related model compounds, Biomolecular Spectroscopy, Wiley & Sons Ltd.,
New York, 1993.
[35] E. Vass, M. Hollosi, F. Besson, R. Buchet, Vibrational spectroscopic detection of
beta- and gamma-turns in synthetic and natural peptides and proteins, Chemical
Reviews 103 (2003) 19171954.
[36] J. Kubelka, T.A. Keiderling, Differentiation of beta-sheet-forming structures: Ab
initio-based simulations of IR absorption and vibrational CD for model peptide
and protein beta-sheets, Journal of the American Chemical Society 123 (2001)
1204812058.
[37] J. Bandekar, Amide modes and protein conformation, Biochimica et Biophysica
Acta 1120 (1992) 123143.
[38] B.I. Baello, P. Pancoska, T.A. Keiderling, Vibrational circular dichroism spectra of
proteins in the amide III region: measurement and correlation of bandshape to
secondary structure, Analytical Biochemistry 250 (1997) 212221.
[39] F.N. Fu, D.B. Deoliveira, W.R. Trumble, H.K. Sarkar, B.R. Singh, Secondary structure
estimation of proteins using the amide-III region of Fourier-transform infrared-
spectroscopy application to analyze calcium binding-induced structural-changes
in calsequestrin, Applied Spectroscopy 48 (1994) 14321441.
[40] S. Peternel, J. Grdadolnik, V. Gaberc-Porekar, R. Komel, Engineering inclusion
bodies for non denaturing extraction of functional proteins, Microbial Cell Factories
7(2008).
[41] S. Yamamoto, J. Kaminský, P. Bouř, Structure and vibrational motion of insulin
from Raman optical activity spectra, Analytical Chemistry 84 (2012) 24402451.
[42] H. Torii, T. Tatsumi, M. Tasumi, Effects of hydration on the structure, vibrational
wavenumbers, vibrational force eld and resonance Raman intensities of
N-methylacetamide, Journal of Raman Spectroscopy 29 (1998) 537546.
[43] S.A. Asher, A.V. Mikhonin, S. Bykov, UV Raman demonstrates that alpha-helical
polyalanine peptides melt to polyproline II conformations, Journal of the Ameri-
can Chemical Society 126 (2004) 84338440.
[44] A.V. Mikhonin, Z. Ahmed, A. Ianoul, S.A. Asher, Assignments and conformational
dependencies of the amide III peptide backbone UV resonance Raman bands,
The Journal of Physical Chemistry. B 108 (2004) 1902019028.
[45] R. Vianello, Z.B. Maksić, Polycyano derivatives of some organic tri- and hexacyclic
molecules are powerful super- and hyperacids in the gas phase and DMSO:
computational study by DFT approach, Journal of Organic Chemistry 75 (2010)
76707681.
[46] D.D. Perrin, B. Dempsey, E.P. Serjeant, pKa Prediction for Organic Acids and Bases,
Chapman and Hall, London; New York, 1981.
[47] J. Makowska, K. Baginska, M. Makowski, A. Jagielska, A. Liwo, F. Kasprzykowski, L.
Chmurzynski, H.A. Scheraga, Assessment of two theoretical methods to estimate
potentiometric titration curves of peptides: comparison with experiment, The
Journal of Physical Chemistry. B 110 (2006) 44514458.
[48] A.I. Arunkumar, T.K.S. Kumar, C. Yu, Specicity of helix-induction by 2,2,2-
triuoroethanol in polypeptides, InternationalJournal of Biological Macromolecules
21 (1997) 223230.
[49] H. Chi, A. Lakhani, A. Roy, M. Nakaema, T.A. Keiderling, Inter-residue coupling
and equilibrium unfolding of PM helical peptides. Vibrational spectra enhanced
with C-13 isotopic labeling, The Journal of Physical Chemistry. B 114 (2010)
1274412753.
[50] K. Xiong, S.A. Asher, Circular dichroism and UV resonance Raman study of the
impact of alcohols on the Gibbs free energy landscape of an alpha-helical peptide,
Biochemistry 49 (2010) 33363342.
[51] M. Jackson, H.H. Mantsch, Beware of proteins in DMSO, Biochimica et Biophysica
Acta 1078 (1991) 231235.
[52] A.M.S. Duarte, C.P.M. van Mierlo, M.A. Hemminga, Molecular dynamics study of
the solvation of an alpha-helical transmembrane peptide by DMSO, The Journal
of Physical Chemistry. B 112 (2008) 86648671.
[53] M.P. Hinderaker, R.T. Raines, An electronic effect on protein structure, Protein Science
12 (2003) 11881194.
[54] P.S. Nerenberg, T. Head-Gordon, Optimizing protein-solvent force elds to reproduce
intrinsic conformational preferences of model peptides, Journal of Chemical Theory
and Computation 7 (2011) 12201230.
[55] P.B. Law, V. Daggett, The relationship between water bridges and the polyproline II
conformation: a large-scale analysis of molecular dynamics simulations and crystal
structures, Protein Engineering, Design & Selection 23 (2010) 2733.
53A. Mirtič, J. Grdadolnik / Biophysical Chemistry 175-176 (2013) 4753
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Measurements of the vibrational Raman optical activity (ROA) spectra of poly(L-lysine) in H2O and D2O solutions, under conditions said to generate model α-helical and unordered conformations, are reported. These provide new insights into the conformational elements actually present. In addition to several signatures characteristic of α-helix, the α-helical state (pH 11.0, 2 °C) of a sample with text-decoration:overlineMw= 26 000 shows a strong sharp positive ROA band at ca. 1340 cm–1, previously assigned to rigid loop structure with local order possibly that of 310-helix, which suggests that the α-helical sections are connected by segments of the corresponding loop. On increasing text-decoration:overlineMw to 268 000 this loop signature grows to dominate the ROA spectrum with a concomitant decrease in the α-helix bands. The unordered state (pH 3.0, 20 °C) shows clear signatures of several distinct conformational elements, including α-helix, β-strand and, possibly, left-handed helix. Under conditions which tend to disrupt secondary structure (5 mol 1–1 NaCl solution heated to 50 °C) the ROA spectrum of the unordered state shows a decrease in the intensity of the signature of the putative left-handed helix and an increase in bands assigned to α-helix.
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We investigated the assignments and the conformational dependencies of the UV resonance Raman bands of the 21-residue mainly alanine peptide (AP) and its isotopically substituted derivatives in both their α-helical and PPII states. We also examined smaller peptides to correlate conformation, hydrogen bonding, and structure. Our vibrational mode analysis confirms the complex nature of the amide III region, which contains many vibrational modes. We assign these bands by interpreting the isotopically induced frequency shifts and the conformational sensitivity of these bands and their temperature dependence. Our assignments of the amide bands in some cases agree, but in other cases challenge previous assignments by Lee and Krimm (Biopolymers 1998, 46, 283−317), Overman and Thomas (Biochemistry 1998, 37, 5654−5665), and Diem et al. (J. Phys. Chem. 1992, 96, 548−554). We see evidence for the partial dehydration of α-helices at elevated temperatures.
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We have examined the UV Raman spectra of a series of amino acids, dipeptides, tripeptides, and the polypeptides poly(L-glutamic acid) and poly(L-lysine) in their random coil, α-helical and β-sheet forms. Our study examines the assignments of the resonance-enhanced amide bands and characterizes their resonance enhancement mechanisms. We have for the first time characterized the conformational dependence of the intense newly assigned band that derives from the overtone of the amide V vibration. We have examined the pH and conformational dependence of the amide band frequencies and Raman cross sections and relate these dependences to changes in the resonant electronic transition frequency and oscillator strength. We use these spectral parameters to monitor the thermal conversion of poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) from the α-helix to β-sheet form and to determine the pH dependence of the transition of PLL to the β-sheet form. We also demonstrate an α-helix-like intermediate of PGA during the transition to the β-sheet conformation. We propose a quantitative relationship between the observed UV Raman spectral cross sections and frequencies and protein secondary structure that will prove useful for conformational studies. These results will serve as the background for analyses of protein secondary structure.