VCD spectroscopic properties of the β-hairpin forming miniprotein CLN025 in various solvents

Article abstract of DOI:10.1002/bip.21356

Electronic and vibrational circular dichroism are often used to determine the secondary structure of proteins, because each secondary structure has a unique spectrum. Little is known about the vibrational circular dichroic spectroscopic features of the β-hairpin. In this study, the VCD spectral features of a decapeptide, YYDPETGTWY (CLN025), which forms a stable β-hairpin that is stabilized by intramolecular weakly polar interactions and hydrogen bonds were determined. Molecular dynamics simulations and ECD spectropolarimetry were used to confirm that CLN025 adopts a β-hairpin in water, TFE, MeOH, and DMSO and to examine differences in the secondary structure, hydrogen bonds, and weakly polar interactions. CLN025 was synthesized by microwave-assisted solid phase peptide synthesis with N^α-Fmoc protected amino acids. The VCD spectra displayed a (-,+,-) pattern with bands at 1640 to 1656 cm^-1, 1667 to 1687 cm^-1, and 1679 to 1686 cm^-1 formed by the overlap of a lower frequency negative couplet and a higher frequency positive couplet. A maximum IR absorbance was observed at 1647 to 1663 cm^-1 with component bands at 1630 cm^-1, 1646 cm^-1, 1658 cm^-1, and 1675 to 1680 cm^-1 that are indicative of the β-sheet, random meander, either random meander or loop and turn, respectively. These results are similar to the results of others, who examined the VCD spectra of β-hairpins formed by ^DPro-Xxx turns and indicated that observed pattern is typical of β-hairpins.

Full text of DOI:10.1002/bip.21356

VCD Spectroscopic Properties of the b-Hairpin Forming Miniprotein
VCD Spectroscopic Properties of the b-Hairpin Forming Miniprotein
CLN025 in Various Solvents
CLN025 in Various Solvents
´
Marcus P. D. Hatfield, Richard F. Murphy, Sandor Lovas
Department of Biomedical Sciences, Creighton University, Omaha, NE 68178
Received 2 October 2009; revised 13 November 2009; accepted 14 November 2009
Published online 23 November 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21356
the results of others, who examined the VCD spectra of b-
hairpins formed by DPro-Xxx turns and indicated that
ABSTRACT:
Electronic and vibrational circular dichroism are often
#
observed pattern is typical of b-hairpins. 2009 Wiley
used to determine the secondary structure of proteins,
Periodicals, Inc. Biopolymers 93: 442–450, 2010.
because each secondary structure has a unique spectrum.
Keywords: CLN025; Chignolin; vibrational circular
Little is known about the vibrational circular dichroic
dichroism; electronic circular dichroism; beta-hairpins;
spectroscopic features of the b-hairpin. In this study, the
miniprotein; molecular dynamics
VCD spectral features of a decapeptide, YYDPETGTWY
(CLN025), which forms a stable b-hairpin that is
stabilized by intramolecular weakly polar interactions
This article was originally published online as an accepted
and hydrogen bonds were determined. Molecular
dynamics simulations and ECD spectropolarimetry were
used to confirm that CLN025 adopts a b-hairpin in
water, TFE, MeOH, and DMSO and to examine
differences in the secondary structure, hydrogen bonds,
and weakly polar interactions. CLN025 was synthesized
by microwave-assisted solid phase peptide synthesis with
Na-Fmoc protected amino acids. The VCD spectra
displayed a (2,1,2) pattern with bands at 1640 to 1656
cm21, 1667 to 1687 cm21, and 1679 to 1686 cm21
formed by the overlap of a lower frequency negative
couplet and a higher frequency positive couplet. A
maximum IR absorbance was observed at 1647 to 1663
preprint. The ‘‘Published Online’’date corresponds to the preprint
version. You can request a copy of the preprint by emailing the
Biopolymers editorial office at biopolymers@wiley.com
INTRODUCTION
odel b-hairpin miniproteins are frequently stud-
ied because they are the simplest form of anti-
parallel b-sheet and often display characteristics
indicative of proteins such as two-state folding.1
b-hairpins are stabilized by cross-strand hydro-
M
gen bonds, disulfide bonds, and weakly polar interactions.2–4
Weakly polar interactions play an important role due to their
strength and ubiquitousness in peptides. Weakly polar inter-
actions can be as strong as hydrogen bonds.5–12 They play an
important role in the stabilization of secondary structure
such as a-helices5 and the tertiary structure of peptides
such as the TC5b8 and APP10,12 miniproteins. Numerous b-
hairpins have been designed to take advantage of the
aromatic–aromatic (Ar–Ar) interactions,13–17 cation–p inter-
actions,16–18 and CH–p interactions.16,17
cm21 with component bands at 1630 cm21, 1646 cm21
,
1658 cm21, and 1675 to 1680 cm21 that are indicative of
the b-sheet, random meander, either random meander or
loop and turn, respectively. These results are similar to
CLN025, Tyr-Tyr-Asp-Pro-Glu-Thr-Gly-Thr-Trp-Tyr,
(Figure 1), is a miniprotein that forms a stable b-hairpin
with a Tm of 708C, which undergoes a reversible cooperative
transition upon thermal denaturation.19 CLN025 is a chigno-
lin variant in which the terminal Gly residues are replaced
with Tyr residues. The b-hairpin has a turn-initiating Asp-Pro
Correspondence to: S. Lovas; e-mail: slovas@creighton.edu
Contract grant sponsor: NIH-INBRE
Contract grant number: P20 RR016469
C
VV
2009 Wiley Periodicals, Inc.
442
Biopolymers Volume 93 / Number 5

VCD Spectroscopic Properties of a b-Hairpin Miniprotein
443
Tyr(OtBu) HMPB ChemMatrix resin and 5M excess of Na-Fmoc
protected amino acids. t-butyl protection was used to protect the re-
active side chains of Asp, Glu, Thr, and Tyr, whereas t-Boc protec-
tion was used for Trp residues. Couplings were performed using
HATU and DIEA at 758C for 10 min with 25 W radiation energy.
Deprotections were performed using 20% (v/v) piperidine and
0.1M HOBt at 758C for 3 min with 35 W radiation energy.
The peptide was cleaved from the resin by stirring in a 95/2.5/2.5
(v/v/v) TFA/TIS/water mixture for 10 min at 08C followed by 110
min at room temperature. The peptide was precipitated with cold
diethyl ether, filtered, dissolved in TFA, and filtered. The filtrate was
evaporated to 1 mL and the peptide was precipitated with cold
diethyl ether and collected by filtration. The peptide was dissolved
in glacial acetic acid which was diluted to 5% acetic acid with nano-
pure water and the solution was lyophilized.
Purification was performed by reverse-phase high-pressure
liquid chromatography using a Gilson dual pump apparatus (Gilson
Inc., Middleton, WI). The peptide was purified on a Phenomenex
Jupiter C18 column (5 lm, 200 mm 3 10 mm; Phenomenex,
Torrance, CA) using an aqueous solvent of 0.1% TFA in water and
an organic solvent of 0.09% TFA in acetonitrile. The peptide was
purified at 308C using a gradient of 10 to 50% organic solvent over
60 min with a flow rate of 4 mL min21. Pure fractions were pooled
and lyophilized. The peptide was identified using a API150EX mass
spectrometer (PE SCIEX, Foster City, CA) and characterized by RP-
HPLC with a Jupiter C18 column (5 lm, 200 mm 3 4.6 mm; Phe-
nomenex, Torrance, CA) using a 3 to 60% organic solvent gradient
FIGURE 1 The backbone and side chains of the crystal structure
of CLN025. Random meander is cyan, b-sheet is red, turn is green,
and the H-bonds are represented by yellow dotted lines.
over 40 min at 1 mL min21
.
sequence20 and is stabilized by an Ar–Ar interaction between
the side chains of Tyr2 and Trp9 and hydrogen bonds between ECD
Asp3 and Thr6, Gly7 and Thr8.19
ECD spectra were recorded using a Jasco J-810 Spectropolarimeter
(JASCO Inc., Easton, MD) by performing 20 scans from 185 to 250
nm at 100 nm min21 scan speed in a 0.05 cm path length quartz
cell. The background spectra of the solvents were subtracted and the
mean residue molar ellipticities were calculated using peptide con-
centrations determined by quantitative RP-HPLC35 with a Jupiter
C18 column (5 lm, 200 mm 3 4.6 mm; Phenomenex, Torrance,
CA). CLN025 was dissolved in either 20 mM KH2PO4-K2HPO4
buffer (pH 7.0), TFE, or MeOH at a final concentration of 100 lM.
CDSSTR36–38 analysis was performed with data set 6 using the
DichroWeb website.38,39–42 The concentration of 100 lM CLN025
was used because at concentrations above 1 mM CLN025 aggregates.
The vibrational circular dichroism spectroscopic proper-
ties of helices,21–24 turns,6,25–27 random meander,23,28 and b-
sheets23,27,29–31 have been studied extensively but the proper-
ties of the b-hairpin have not. Previous studies of the VCD
spectroscopic properties of b-hairpins were of peptides with
DPro-Xxx turns or head-to-tail cyclization to stabilize the
structure in chloroform or aqueous solvents rather than sol-
vents such as TFE, MeOH, or DMSO.32–34 Many of the
designed b-hairpins aggregate at higher concentrations in
water. This hampers experiments to examine VCD spectra of
b-hairpins containing only L-amino acids. In this study, the
VCD spectroscopic properties of CLN025, which has been
VCD
CLN025 was dissolved in 0.1M DCl in D2O and lyophilized three
shown to form a stable b-hairpin containing only naturally times before VCD measurements to remove all trifluoroacetic acid
salts and perform a H/D exchange on the backbone of the peptide.
occurring amino acids, in aqueous solution and in different or-
All samples for VCD measurement were prepared to have a concen-
ganic solvents (TFE, MeOH, and DMSO) were determined.
tration of 20 mg mL21 CLN025. Buffer solutions were prepared by
Molecular dynamics (MD) simulations and ECD were used to
dissolving 4 mg of CLN025 in 10 lL DMSO-d6 and gradually dilut-
confirm the b-hairpin conformation of CLN025 in the solvents.
ing this solution to 5% DMSO-d6 with 20 mM KD2PO4-K2DPO4
buffer (pD 7.4). All other solutions were prepared by dissolving 2
mg CLN025 in either TFE-d, MeOH-d4 or DMSO-d6.
VCD spectra were recorded using a dual PEM BOMEM Biotools
METHODS
Chiralir Fourier Transform VCD Spectropolarimeter (BioTools Inc.,
Jupiter, FL). Either 4- or 8-block measurements consisting of 4500
CLN025 Synthesis and Purification
CLN025 was synthesized with a CEM Liberty microwave peptide scans for VCD and 225 scans for IR absorption at 8 cm21 resolution
synthesizer (CEM, Matthews, NC) at a 0.1 mmol scale using a H- were performed in a 75 lm pathlength CaF2 cell. 8 blocks were
Biopolymers

444
Hatfield, Murphy, and Lovas
recorded for VCD measurements of CLN025 in buffer, TFE-d,
MeOH-d4, and DMSO-d6 at 208C, whereas 4 blocks were recorded
for VCD measurements in buffer at 08C, 208C, and 608C. Spectra of
the blocks were averaged and the background spectra of the solvent
and water vapor were subtracted. Spectra were set to 0 at 1800 cm21
and normalized43 by dividing each data point by the maximum IR
absorbance in the amide I0 region. The spectra were normalized to
remove the effect of small differences in the sample concentration
on the intensity of the spectra.
Maximum-entropy deconvolution and peak fitting analysis of
the FTIR spectra were performed with the PROTA v 3.0 software
package (BioTools Inc., Jupiter, FL) using the tyrosyl CꢀꢀC ring
vibration band at 1515 cm21 as the representative peak shape,
which has been found to be a good marker due to its sharpness, nar-
row width, and discrete location.44 Peak-fitting analysis is not con-
clusive, because it produces no unique band assignment, molar
absorptivities of all conformations are assumed to be equal and con-
clusions can be subjective.45 The analysis performed by the PROTA
software package, however, is less subjective, by using Bayesian
derivatives and maximum likelihood functions to generate a list of
peaks that meet acceptable signal to noise criteria and determines
the areas of the individual peaks with the Levenberg-Marquardt
maximum likelihood algorithm.
FIGURE 2 The ECD spectra of 100 lM CLN025 in 20 mM potas-
sium phosphate buffer at 08C (violet), 208C (blue), 408C (green),
608C (orange), and 808C (red) and cooled from 808C to 08C
(black).
to those measured by Honda et al.,19 except their spectra
showed a more intense negative band at 197 nm. The spectra
indicate that the peptide has b-sheet, Type II b-turn and ran-
dom meander, as well as an aromatic couplet caused by the
Ar–Ar interactions.
Molecular Dynamics Simulations
MD simulations were performed using the crystal structure of
CLN02519 with the GROMACS 3.2.1 software package46,47 using the
GROMOS 96 force field with the 53a6 parameter set.48,49 CLN025
was solvated in either 1438 SPC50 water, 302 TFE, 1866 MeOH, or
286 DMSO molecules in a 45.61 nm3 dodecahedral box with one
chloride and three sodium ions to neutralize the system. The
MeOH, TFE, and DMSO models used were optimized for the 53a6
parameter set49 and the experimentally determined compressibilities
(4.92 3 1025 bar21, 4.50 3 1025 bar21, 4.45 3 1025 bar21, 1.22 3
1024 bar21, 1.29 3 1024 bar21, and 4.50 3 1025 bar21) and dielec-
tric constants (86, 78, 68, 27, 32, 47) were used for the simulations
of CLN025 in SPC water at 278, 300, and 333 K and TFE, MeOH,
and DMSO at 300 K, respectively.51 The solvated structures were
energy-minimized using steepest descent.52 An NVT simulation of
the positionally restrained peptide was performed for 100 ps at 300
K (278C) followed by a 200.1 ns NPT simulation at 300 K and 1 bar
using Berendsen coupling.53 All bonds were constrained54 with
Shake55 in the TFE simulation and Lincs54 in the other simulations.
In addition, MD simulations were performed in SPC water at 278 K
(58C) and 333 K (608C).
As the temperature increases, the intensity and position of
the positive band of the aromatic couplet shifts from 225 to
230 nm and at some temperatures more than one couplet is
apparent most likely due to contributions from the Tyr2–
Trp9 and Tyr1–Tyr10 Ar–Ar interactions as well as the Tyr1–
Tyr9 interaction at higher temperatures. This provides fur-
ther evidence that using the aromatic couplet as a measure of
folding is not accurate since, in peptides containing more
than two aromatic rings, the interactions could shift causing
a shift in the aromatic couplet that is not indicative of the
overall fold.
As the temperature increases above 608C, a positive band
at 212 nm becomes visible. This is indicative of random me-
ander, indicating that the b-hairpin is destabilized. The
increase in random meander content most likely results from
a loss of b-sheet, which would explain why the intensity of
the 195 nm band does not change. The spectra, however, still
suggests b-hairpin conformation and the aromatic couplet
indicates that the ends of the peptide remain in close prox-
imity. The ECD spectra of the peptide at 08C and the peptide
cooled from 808C to 08C are similar, indicating reversible
unfolding.
MD simulations were analyzed by examining the middle struc-
ture of the largest cluster determined by the backbone RMSD of
structures sampled every 10 ps along the trajectory. Secondary
structure of CLN025 was analyzed by the DSSP method.56
RESULTS AND DISCUSSION
The ECD spectra of CLN025 in buffer, TFE, and MeOH
are shown in Figure 3. The spectra have similar characteris-
tics, but the peaks in the spectrum in TFE are blue-shifted 2
to 3 nm, whereas in MeOH, they are red-shifted 1 to 2 nm
from the positions in the spectrum of the peptide in buffer.
ECD Spectroscopy
The ECD spectra of 100 lM CLN025 in 20 mM potassium
phosphate buffer (pH 7.0) from 0 to 808C and cooled from
808C to 08C are shown in Figure 2. These spectra are similar
Biopolymers

VCD Spectroscopic Properties of a b-Hairpin Miniprotein
445
FIGURE 3 The ECD spectra of 100 lM CLN025 in 20 mM potas-
sium phosphate buffer (black), TFE (red), and MeOH (blue) at
208C.
This is most likely due to solvent effects including differing
degrees of hydration of individual amide groups45,57–59 and
differences in the dipolar character of the solvent.60–63 The
aromatic couplet in TFE is less intense due to the tendency of
high concentrations of TFE to weaken the interactions
between hydrophobic side chains.64,65 The higher intensity of
the aromatic couplet in MeOH suggests a stronger Ar–Ar
interaction. Because of the overlap of the aromatic couplet
with signals conveying information about the secondary
structure,13,66 it is difficult to determine if small changes in
the conformation of the peptide occur in the different sol-
vents. CDSSTR analysis (Table I), however, suggests that the
peptide is 46% sheet, 23% turn, and 31% random meander
in buffer. In TFE and MeOH, the sheet component increases
to 56% and 59%, respectively, whereas the random meander
component decreases to 20% in TFE and the turn compo-
nent decreases to 13% in MeOH.
FIGURE 4 Normalized VCD (top) and IR absorption (bottom)
spectra of 20 mg mL21 CLN025 in 5% (v/v) DMSO-d6 in 20 mM
deuterated potassium phosphate buffer (black), TFE-d (red),
MeOH-d4 (blue), and DMSO-d6 (green). The deconvoluted absorp-
tion spectra are shown by the thin curves.
The ECD spectra of CLN025 are similar at all tempera-
tures and in the different solvents indicating no significant
conformational change in the structure of CLN025 upon
heating and in either TFE or MeOH. This demonstrates the
stability of the b-hairpin in CLN025. The small changes in
the ECD spectra of CLN025 indicate that at high tempera-
tures, the amount of random meander increases and the Ar–
Ar interactions decrease. In TFE, CLN025 displays a higher
b-sheet content and a decrease in the amount of random me-
ander. In MeOH, the b-sheet and random meander content
of CLN025 increases and the turn content decreases.
Table I Structural Content (%) of CLN025 in Different
Solvents as Determined by the Analysis of ECD Spectra
Using the CDSSTR Method
Solventa
Strand
Turn
Random Meander
VCD Spectroscopy
The VCD and FTIR spectra of CLN025 in various solvents
are shown in Figure 4 and the position of the VCD bands are
listed in Table II. The IR spectrum of CLN025 in buffer has a
maximum absorbance at 1647 cm21 with shoulders at 1675
cm21, 1646 cm21, 1630 cm21, and 1612 cm21 in the decon-
Buffer
TFE
MeOH
46
56
59
23
24
13
31
20
28
a
See methods.
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446
Hatfield, Murphy, and Lovas
Table II Position of Bands (cm21) in the VCD
Spectra of CLN025
turn structures. The signals at 1646 cm21 and 1658 cm21 are
due either to random meander or loop conformations. The
major signal shifts from 1646 cm21 to 1658 cm21 when the
peptide is in nonaqueous solutions, which causes the shift of
the negative VCD couplet in these solvents. The signal at
1675 cm21 is due either to the turn or the high-frequency ab-
sorbance of b-sheet23,44 although turn is more likely due to
the high signal of this peak in DMSO. The shift of the 1675
cm21 band to 1680 cm21 in TFE is the likely cause of the
shift in the positive couplet in the VCD spectrum. The 1705
Solvent
2 Band
1 Band
2 Band
Buffer
TFE
1679
1686
1679
1667
1671
1671
1687
1640
1647
1647
1656
MeOH
DMSO
voluted spectra, which are indicative of turn, random mean- cm21 peak in TFE and MeOH is most likely due to the
der, b-sheet, and tertiary amide or tyrosine side-chain vibra- glutamic acid side chain, indicating that this side chain is not
tion, respectively.15,23,27,44,67 The VCD spectra show a shielded. The 1712 cm21 band seen in buffer and especially
(2,1,2) pattern, formed by the overlap of the positive in DMSO, where it accounts for 25% of the amide I0 signal, is
bands of a negative and positive couplet, with bands at 1640 due either to the aspartic acid side chain or a change in struc-
cm21, 1667 cm21, and 1679 cm21, respectively. The first ture. The aspartic acid side chain provides a better explana-
couplet, centered at 1653 cm21, is indicative of random me- tion due to the lack of a VCD signal in this region and
ander and may indicate a local left-handed twist,28 whereas because of the shift in the orientation of the side chain in the
the second couplet, centered at 1673 cm21, is indicative of a structure of CLN025 in DMSO (Figure 6F).
Type II0 b-turn.25,68
The CDSSTR analysis of the ECD spectra (Table I) and
The IR absorbance spectra of the peptide in TFE and the peak fitting analysis of the FTIR spectra (Table III) show
MeOH are similar to the IR spectrum of the peptide in similarities in the differences of b-sheet, turn, and random
buffer, except the maximum absorbance is shifted to 1655 meander content in different solvents. Each analysis indicates
cm21. The peaks of the VCD spectra of the peptide in TFE that in TFE the b-sheet content of CLN025 increases and the
and MeOH are also shifted from positions in buffer with the random meander content decreases. In MeOH, both analyses
negative band shifted to 1647 cm21 and the positive band to indicate a loss of turn content but the peak-fitting analysis
1671 cm21. In MeOH, the second negative VCD band is at indicates a slight loss of b-sheet and an increase in the ran-
the same location as the second negative VCD band of the dom meander content of CLN025. This discrepancy between
peptide in buffer, whereas in TFE, this band is shifted up to the results of the two analyses is most probably due to the in-
1686 cm21 which indicates a change in the turn structure of terference of the signal from the aromatic couplet in the ECD
CLN025 in TFE.
spectra. Even though the amount of turn in the peptide in
In DMSO, the maximum IR absorbance is shifted to 1663
cm21 and the 1612 cm21 absorbance peak is resolved due to
a reduced shoulder around 1630 cm21, indicating a loss of b-
sheet. The peaks at 1706 cm21 and 1712 cm21 are indicative
of the Glu and Asp side chains in deuterated solvents, respec-
tively,44 or a change in the turn-like structure of the peptide.
The VCD of CLN025 in DMSO shows only a negative cou-
plet with a negative component at 1656 cm21 and positive
component at 1687 cm21, attributed to a greater relaxation
of the turn that causes the second couplet to be obscured by
the first couplet.
Table III The Areas of the Component Peaks in the
Deconvoluted FTIR Spectrum at the Amide I0 Region of CLN025
as a Percentage of the Total Area of the Amide I0 Peak
Wavenumber/
cm21
Buffer TFE MeOH DMSO
5
Assignment
Turn
1722
1712
1705
1696
1680
1675
1658
5
24
Turn or Asp
Turn or Glu
Turn
6
8
2
2
Turn
Turn or b-sheet
Random
Peak-fitting analysis of the FTIR spectra, shown in Table
III, indicates that the 1612 cm21 signal contributes equally in
the different solvents, which indicates that the environment
around the prolyl residue does not change significantly. The
1630 cm21 signal indicates that CLN025 is 16%, 24%, and
11% b-sheet in buffer, TFE, and MeOH, respectively, whereas
a signal accounting for 3% of the amide I0 area in DMSO is
5
27
5
57
25
45
53
meander or loop
Random meander
1646
1630
1612
40
16
4
22
11
3
24
4
3a b-sheet
3
Pro
a
May be due to the low-frequency absorbance of turn structure rather
due either to b-sheet or the low-frequency absorbance of than b-sheet.
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VCD Spectroscopic Properties of a b-Hairpin Miniprotein
447
MeOH decreases, the position of the second couplet and the
relative contributions of the amide bands at 1646 cm21 and
1658 cm21 suggest that the turn in the peptide in MeOH is
more similar to that in buffer than the turn in TFE is to that
in buffer. Although the CDSSTR analysis of the ECD spectra
of CLN025 in buffer indicates that the b-hairpin is 46%
sheet, 23% turn, and 31% random meander, the peak fitting
analysis of the VCD indicates that the b-hairpin is 16% sheet,
12% turn, and 67% random meander. The major differences
between the overall amounts of the b-sheet, turn, and ran-
dom meander assigned by the two analyses could be due to
the interference of the aromatic couplet in the ECD spectra
and the faulty assumption in peak fitting analysis that the
molar absorbtivities of all conformations are equal.45
The VCD spectra of CLN025 in buffer at 58C, 208C, and
608C, shown in Figure 5, are similar indicating that the b-
hairpin is stable. The less pronounced shoulder at 1630 cm21
in the IR absorption spectrum indicates that the amount of
b-sheet content decreases at 608C.
The analysis indicates that the VCD spectra of CLN025
and b-hairpins in general consist of overlapping negative and
positive couplets. The negative couplet is due to random me-
ander or loop structure, whereas the higher frequency posi-
tive couplet is due to the turn. This produces a (2,1,2) pat-
tern with bands at 1640 to 1656 cm21, 1667 to 1687 cm21
,
and 1679 to 1686 cm21. The b-sheet component of the VCD
spectra is not visible due to its low intensity29 and overlap
with the first negative band (1640 to 1656 cm21). The b-
sheet, however, might contribute to the greater width of the
first negative band. The IR absorption spectra show a broad
amide I0 band with a maximum at 1647 to 1663 cm21 and
bands at 1630 cm21, 1646 cm21, 1658 cm21, and 1675 to
FIGURE 5 Normalized VCD (top) and IR absorption (bottom)
spectra of 20 mg mL21 CLN025 in 5% (v/v) DMSO-d6 in 20 mM
deuterated potassium phosphate buffer at 58C (black), 208C (blue),
and 608C (red).
1680 cm21 which are indicative of the b-sheet, random me- to 1680 cm21 in the VCD spectra and a maximum IR
ander, random meander, or loop and turn, respectively. The absorbance at 1638 cm21. These results are similar to those
shifts in the peaks of the VCD and IR absorption spectra of here, except that the maximum IR absorbance was 9 cm21
the peptides in the different solvents is likely due to solvent lower. This is most likely due to a greater amount of b-sheet
effects, which have been shown to alter the spectral shapes of structure in dodecapeptide. Hilario et al.34 examined spectra
D
b-hairpins due to differing degrees of hydration of the indi- of 4 to 16 residue peptides with a Pro-Gly turn in aqueous
vidual amide groups45,57–59 and differences in the dipolar solution and found a negative couplet with negative band at
character of the solvent.60–63
1635 to 1645 cm21 and positive band at 1660 to 1670 cm21
The results are similar to the results found by other in the VCD spectra and a maximum IR absorbance at 1635
groups for b-hairpins that have DPro-Xxx turns. Zhao et al.32 to 1645 cm21
examined the spectra of numerous octapeptides with a DPro-
Xxx turn in chloroform and found a (2,1,2,1) pattern of
.
VCD peaks at 1643 to 1659 cm21, 1655 to 1670 cm21, 1670 MD Simulations
to 1678 cm21, and 1690 to 1697 cm21, respectively, with a Cluster analysis, based on the backbone RMSD of the pep-
maximum IR absorbance at 1690 to 1697 cm21. Hilario tide, generated 8, 15, 76, 3, 19, and 6 clusters for CLN025 in
et al.33 examined the VCD spectra of a dodecapeptide with a H2O at 58C, H2O at 278C, H2O at 608C, TFE at 278C, MeOH
DPro-Gly turn in aqueous solution and found a negative cou- at 278C, and DMSO at 278C, respectively. The middle struc-
plet with negative band at 1638 cm21, positive band at 1660 tures of the largest clusters are seen in Figure 6 and account
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448
Hatfield, Murphy, and Lovas
FIGURE 6 The backbone and side chains of the middle structure of the most populated cluster of
trajectories of MD simulations of CLN025 in A, H2O 58C; B, H2O 278C; C, H2O 608C; D, TFE; E,
MeOH; and F, DMSO. Random meander is cyan, b-sheet is red, turn is green, and the H-bonds are
yellow dotted lines.
for 92.3%, 94.4%, 72%, 99.8%, 83.9%, and 98.2%, respec- ticipate in more hydrogen bonds. In MeOH, the peptide
tively, of the 20,000 structures sampled. The small number of maintains a tight turn though shortened and the Gly7 resi-
clusters and the large contribution of the largest cluster indi- due is assigned to the b-sheet region. In H2O at 608C, the
cate the CLN025 forms a stable structure in all environments turn is flattened causing the Gly7 residue to rotate and the
studied. The middle structures indicate that the b-hairpin C-terminal strand changes conformation by elongation. This
conformation is maintained in all environments but the turn causes the hydrogen bond pattern to change and the Trp9
is relaxed in TFE and DMSO showing a random meander residue to move further away from the Tyr2 residue breaking
from Pro4 to Gly7 in TFE and from Asp3 to Gly7 in DMSO. the Ar–Ar interaction and forming a new Ar–Ar interaction
In DMSO, the Asp3 side chain moves closer to the opposite with the Tyr1 residue.
strand and rotates so that the oxygen atoms are pointing to- DSSP analysis (Figure 7) further confirms that the b-hair-
ward the turn and strand. This allows the side chain to par- pin structure of CLN025 in all environments studied has a
FIGURE 7 DSSP analysis of the trajectories of simulations of CLN025 in A, H2O 58C; B, H2O
278C; C, H2O 608C; D, TFE; E, MeOH; and F, DMSO. Random meander is black, b-sheet is red, b-
bridge is white, bend is blue, and turn is green.
Biopolymers

VCD Spectroscopic Properties of a b-Hairpin Miniprotein
449
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Pro-Glu-Thr-Gly turn flanked by two residues on each side
in a b-sheet or b-bridge conformation. At 608C, Trp9
changes from being in a b-sheet conformation to a random
meander conformation. In DMSO, the b-sheet structure of
the peptide is diminished significantly. Tyr2 and Trp9 form a
b-bridge, the turn is relaxed and the Asp3, Gly7, and Thr8
residues are in a random meander conformation.
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The MD simulations confirm the results of the ECD and
VCD spectral analyses. CLN025 maintained a b-hairpin con-
formation in all environments studied. In TFE, the turn is
relaxed as shown by the shift in the second couplet of the
VCD spectra and its corresponding amide I0 bands in the IR
spectra as well as by the middle structures of the most popu-
lated cluster from the MD simulations and the DSSP analysis.
In MeOH, the turn in CLN025 is shortened, as seen by the
loss of turn content from the CDSSTR analysis of the ECD
spectra, the results of the peak-fitting analysis of the VCD
spectra and the middle structure of the peptide in MD analy-
ses. In MeOH, however, the peptide maintains the same tight
conformation as the peptide in water as indicated by the
position of the second couplet in the VCD spectra and
the assignment of the turn region in the middle structures of
the peptide. In DMSO, the turn is relaxed and is mostly
random meander as seen by the lack of an evident second
couplet in the VCD spectra as well as by the DSSP analysis
and the middle MD structures of the peptide.
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CONCLUSIONS
The VCD spectral properties of a 10 residue miniprotein,
CLN025, that forms a stable b-hairpin, with only naturally
occurring amino acids and without D-amino acids or cycliza-
tion to stabilize the b-hairpin, are reported. ECD, VCD, and
MD simulations confirm that CLN025 adopts a stable b-
hairpin conformation in the environments studied. The VCD
spectra exhibit a (2,1,2) pattern with bands at 1640 to
1656 cm21, 1667 to 1687 cm21, and 1679 to 1686 cm21. A
maximum IR absorbance was observed at 1647 to 1663 cm21
with bands at 1630 cm21, 1646 cm21, 1658 cm21, and 1675
to 1680 cm21 in the deconvoluted spectra, indicating b-
sheet, random meander, random meander, or loop and turn,
respectively. These results are similar to those reported for
peptides in which DPro was incorporated to nucleate the b-
turn in the hairpin and suggest that the VCD and IR absorb-
ance spectra reported here are typical of b-hairpins.
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