A photocontrolled β-hairpin peptide

Article abstract of DOI:10.1002/chem.200500986

β-Hairpins constitute the smallest β-type structures in peptides and proteins. The development of highly stable, yet monomeric β-hairpins based on the tryptophan zipper motif was therefore a remarkable success [A. G. Cochran, N. J. Skelton, M. A. Starovasnik, Proc. Natl. Acad. Sci USA 2001, 98, 5578-5583]. We have been able to design, synthesize and characterize a hairpin based on this motif which incorporates an azobenzene-based photoswitch, that allows for time-resolved folding studies of β-structures with unprecedented time resolution. At room temperature the trans-azo isomer exhibits a mostly disordered structure; however, light-induced isomerization to the cis-azo form leads to a predominantly extended and parallel conformation of the two peptide parts, which are linked by the novel photoswitch, [3-(3-aminomethyl)-phenylazo] phenylacetic acid (AMPP). While in the original sequence the dipeptide Asn-Gly forms a type I′ β-turn which connects the two strands of the hairpin, this role is adopted by the AMPP chromophore in our photoresponsive β-hairpin that can apparently act as a β I′-turn mimetic. The β-hairpin structure was determined and confirmed by NMR spectroscopy, but the folding process can be monitored by pronounced changes in the CD, IR and fluorescence spectra. Finally, incorporation of the structurally and functionally important β-hairpin motif into proteins by chemical ligation might allow for the photocontrol of protein structures and/or functions.

Full text of DOI:10.1002/chem.200500986

DOI: 10.1002/chem.200500986
A Photocontrolled b-Hairpin Peptide
Shou-Liang Dong,[a, c, e] Markus Lçweneck,[a, e] Tobias E. Schrader,[b]
Wolfgang J. Schreier,[b] Wolfgang Zinth,[b] Luis Moroder,[a] and Christian Renner*[a, d]
Abstract: b-Hairpins constitute the
smallest b-type structures in peptides
and proteins. The development of
highly stable, yet monomeric b-hairpins
based on the tryptophan zipper motif
was therefore a remarkable success
[A. G. Cochran, N. J. Skelton, M. A.
Starovasnik, Proc. Natl. Acad. Sci USA
2001, 98, 5578–5583]. We have been
able to design, synthesize and charac-
terize a hairpin based on this motif
which incorporates an azobenzene-
based photoswitch, that allows for
time-resolved folding studies of b-
structures with unprecedented time res-
olution. At room temperature the
trans-azo isomer exhibits a mostly dis-
ordered structure; however, light-in-
duced isomerization to the cis-azo form
leads to a predominantly extended and
parallel conformation of the two pep-
tide parts, which are linked by the
novel photoswitch, [3-(3-aminomethyl)-
phenylazo]phenylacetic acid (AMPP).
While in the original sequence the di-
peptide Asn-Gly forms a type I’ b-turn
which connects the two strands of the
hairpin, this role is adopted by the
AMPP chromophore in our photores-
ponsive b-hairpin that can apparently
act as a b I’-turn mimetic. The b-hair-
pin structure was determined and con-
firmed by NMR spectroscopy, but the
folding process can be monitored by
pronounced changes in the CD, IR and
fluorescence spectra. Finally, incorpo-
ration of the structurally and function-
ally important b-hairpin motif into pro-
teins by chemical ligation might allow
for the photocontrol of protein struc-
tures and/or functions.
Keywords: azobenzene
·
NMR
spectroscopy · peptides · photo-
chemistry · protein folding
Introduction
[a] Dr. S.-L. Dong, Dipl.-Chem. M. Lçweneck, Prof. Dr. L. Moroder,
Dr. C. Renner
Of the two major structural elements of proteins, a-helices
have been thoroughly investigated for many decades, where-
as defined models of b-sheets have been developed only in
recent years.[1] In contrast to helices, where local (i,i+3 ) or
(i,i+4) hydrogen bonds confer stability, b-strands require a
structural context for stabilization. Tertiary structure is,
therefore, the crucial prerequisite for the b-extended secon-
dary structure. The smallest structural motif consisting of
short b-strands is the b-hairpin motif, where two antiparallel
strands are linked by a turn or small loop. In proteins b-hair-
pins often constitute binding epitopes and are involved in
protein–protein or protein–DNA interaction.[2] While model
a-helices have been studied extensively for decades, models
for b-structure are less accessible. Besides the requirement
for at least two b-strands, peptides with preference for an
extended conformation are usually prone to aggregation.
Additionally, small b-structures such as the b-hairpin are
only marginally stable and more difficult to characterize
spectroscopically than a-helices.[1] The development of
highly stable, yet monomeric b-hairpins based on, for exam-
ple, a designed tryptophan zipper motif[3] has been a major
Max-Planck-Institut für Biochemie
Am Klopferspitz 18, 82152 Martinsried (Germany)
Fax : (+49)89-8578-2847
E-mail: renner@biochem.mpg.de
christian.renner@ntu.ac.uk
[b] Dipl.-Phys. T. E. Schrader, W. J. Schreier, Prof. Dr. W. Zinth
Lehrstuhl für Biomolekulare Optik
Ludwig-Maximilians-Universität
Oettingenstrasse 67, 80538 München (Germany)
[c] Dr. S.-L. Dong
Present address:
School of Life Science, Lanzhou University
222 Tianshui South Road, Lanzhou 730000 (China)
[d] Dr. C. Renner
Present address:
School of Biomedical and Natural Sciences
Nottingham Trent University
Clifton Lane, Nottingham NG11 8NS (UK)
[e] Dr. S.-L. Dong, Dipl.-Chem. M. Lçweneck
Both authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author: Four tables con-
taining NMR resonance assignments and distance constraints for both
cis-azo and trans-azo isomer of the AMPP-peptide.
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FULL PAPER
precoated silica gel 60 F254 plates (Merck, Germany) and visualized
using UV irradiation (254 nm). Flash chromatography was performed on
silica gel 60 (Merck, Germany). Analytical RP-HPLC was carried out
with Waters equipments on a XTerra C8 150”3.9 mm column (Waters,
Germany) using a linear gradient of acetonitrile/2% H3PO4 from 5:95 to
90:10 in 15 min at a flow rate of 1.5 mLminꢀ1. ESI-MS was recorded on a
Perkin–Elmer SCIEX API 165 spectrometer.
step forward and allowed successful experimental and theo-
retical folding studies.[4] Molecular dynamics simulations
using the folding@home approach[5] found overall folding
rates in good agreement with experimental time-resolved
temperature jump experiments.[4]
Following our previous strategy of combining small pro-
tein motifs with an azobenzene photoswitch,[6] the aim of
the present study was the design of a tryptophan zipper-like
b-hairpin with a turn containing azobenzene (Figure 1). The
azobenzene chromophore offers the possibility to control
the hairpin structure and initiate a folding or unfolding tran-
sition. High isomerization yield, remarkable photostability
and ultra-fast kinetics (few ps) make azobenzene an ideal
trigger for the use in time-resolved ultra-fast pump-probe
spectroscopy of peptides. Small, well-defined model systems
with an ultrafast trigger allow detailed folding studies to be
carried out both experimentally and theoretically on the
same molecules. Only the comparison of simulation and ex-
periment permits assessment and improvement of the theo-
retical description on one side and a detailed interpretation
of the observed rate constants on the other hand. Recently,
we had been able to introduce the first model where ultra-
fast conformational dynamics of a peptide could be investi-
gated by both femtosecond absorption spectroscopy and all-
atom molecular mechanics simulations.[7]
(3-Aminobenzyl)carbamic 9H-fluoren-9-yl methyl ester (1): A solution of
Fmoc-OSu (14.9 g, 44 mmol) in acetonitrile (100 mL) was added drop-
wise over a period of 30 min at room temperature to a stirred solution of
(3-amino)benzylamine (44 mmol, 5 mL) and Et3N (6.1 mL, 44 mmol) in
acetonitrile/DMF 10:1 (55 mL). After the reaction mixture was stirred
for 1 h, the product was precipitated with water, filtered off and washed
with methyl tert-butyl ether/trifluoroethanol 1:1 to give compound 1 as a
white solid (8.73g, 58%); analytical data were identical to those reported
in ref. [8].
3-Nitrosophenylacetic acid (2): NH4Cl (0.48 g, 9.0 mmol) and Zn powder
(0.88 g, 13.5 mmol) were added portionwise at room temperature to a so-
lution of 3-nitrophenylacetic acid (0.91 g, 5.0 mmol) in 2-methoxyethanol
(25 mL). After 45 min stirring, the mixture was cooled to 08C on an ice
bath, and a solution of FeCl3·6H2O (2.12 g, 75 mmol) in ethanol/water
5:1 (30 mL) was added dropwise. After the reaction mixture was stirred
for 1 h, the mixture was extracted, diluted with diethyl ether and the or-
ganic phase was washed three times with water and dried over Na2SO4.
Flash column chromatography with ethyl acetate/hexane/acetic acid
100:5:1 furnished 2 (0.39 g, 47%) as a brownish solid which was used
without further purification.
3-[(3-N-Fmoc-aminomethyl)phenylazo]phenylacetic acid (3, Fmoc-
AMPP-OH): Compound 1 (0.78 g, 2.25 mmol) was added portionwise to
a solution of 2 (0.76 g, 4.55 mmol) in acetic acid (5 mL), and the resulting
mixture stirred for 12 h at room temperature. The precipitate was filtered
off and washed with diethyl ether to give the compound 3 (0.57 g, 51%)
as
a bright orange solid; HPLC: tr =7.65 min (cis isomer) and tr =
8.65 min (trans isomer); analytical data were identical to those reported
in ref. [8].
H-Ser-Trp-Thr-Trp-Glu-AMPP-Lys-Trp-Thr-Trp-Lys-NH2: The peptide
containing the AMPP building block was synthesized on a 0.25 mmol
scale by standard protocols of Fmoc chemistry on an automated peptide
synthesizer (PerSeptive Biosystems, Pioneer) with TentaGelS RAM resin
(Rapp Polymere GmbH, Tübingen, Germany, 0.28 mmolgꢀ1) as solid
support. The Fmoc-protected amino acids (Iris Biotech GmbH, Mark-
tredwitz, Germany) were coupled in four-fold excess by HOBt/HBTU/
DIEA 1:1:2 in NMP, while coupling of Fmoc-AMPP-OH proceeded man-
ually under identical conditions. Fmoc cleavage was performed with 20%
piperidine in NMP (3”1min and 1”15 min). Cleavage from the resin
and deprotection were carried out with TFA/water (95:5) in 2 h. The
crude peptide was purified by RP-HPLC on a preparative column (VP
250/21 Nucleosil 100–5 C8, Macherey and Nagel, Düren, Germany) by
eluting with a linear gradient from 20 to 65% acetonitrile in 0.1% aque-
ous TFA at a flow rate of 10 mLminꢀ1. Fractions containing homogene-
ous material were pooled and lyophilized (59 mg, 16%); HPLC: tr =
4.9 min (cis isomer) and 5.3min ( trans isomer); ESI-MS: m/z: 845.0
[M+2H]2+; Mr: calcd for C87H106N20O16: 1688.0; the 1H NMR (500 MHz
in CD3OH) spectra for the cis- and trans isomer were consistent with the
assigned structure (see Tables S1 and S2 of the Supporting Information).
Figure 1. Structure of the b-hairpin peptide with the AMPP chromo-
phore.
Herein we present the synthesis and characterization of a
b-hairpin based on the tryptophan zipper motif which incor-
porates an azobenzene-based photoswitch and thus allows
for time-resolved folding studies of b-structures with unpre-
cedented temporal resolution. During the preparation of
this manuscript a b-hairpin similar to ours has been publish-
ed.[8] While both hairpin models share the same chromo-
phore proposed by Kräutler et al.[9] the tryptophan zipper
motif applied in our model compound resulted in good solu-
bility of both the cis-azo and trans-azo isomers; this behav-
ior allowed a structural characterization, whereas the amino
acid sequence chosen by Hilvert and colleagues led to insol-
ubility of the trans-azo isomer[8] possibly compromising its
use in folding studies.
Circular dichroism: CD spectra in the 190–250 nm range were recorded
on a JASCO J-715 spectropolarimeter equipped with a thermostated cell
holder. Spectra were obtained at peptide concentrations between 25 and
90 mm employing quartz cells of 1 or 2 mm optical path length and are re-
ported in terms of molar ellipticity per residue ([V]R). The concentrations
were determined by UV absorption of the Trp residues and the AMPP
chromophore. The CD spectra of the cis-azo isomer were recorded after
irradiation at 360 nm until the photostationary state was reached (~80%
cis-azo isomer as determined by 1H NMR experiments upon irradiation
of a trans-azo isomer under identical conditions). The CD spectrum of
the trans-azo isomer was recorded after thermal relaxation of the sample
in the dark.
Experimental Section
Synthesis of the AMPP chromophore
Materials: All commercially available reagents and solvents were used as
received. Reactions were monitored by thin-layer chromatography on
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1115

C. Renner et al.
Photoisomerization: A xenon lamp 450 XBO (Osram, München) was
used for irradiation at 360 or 430 nm (filter from Itos, Mainz) with a light
intensity of ~20 mW in all experiments except for IR spectroscopy (see
below). The photostationary states are reached after irradiation for ca
10 min at the required 360 and 430 nm wavelengths, respectively. A maxi-
mum of cis- to trans-azo ratios of 80:20 and 20:80 are obtained at the
photostationary states as determined from integrals of well-separated res-
lations, because they were mutually exclusive indicating fast conforma-
tional averaging (with rotation about the connecting bond) on the NMR
time scale of milliseconds. The final trans-azo structural ensemble exhib-
ited no significant or systematic violations of NMR constraints, but dis-
played two distinct conformational families differing in the relative orien-
tation of the two peptide strands and the AMPP chromophore.
Molecular modeling: All calculations were performed with the DISCOV-
ER program (Accelrys, San Diego, CA) on Silicon Graphics O2 R5000
computers. The force field CVFF with a time step of 1 fs and a cutoff of
12 Š for the non-bonded interactions was employed for all calculations.
1
onances of both isomers in 1D H NMR spectra.
Fluorescence spectroscopy: Emission spectra in the 300–400 nm range
were recorded upon excitation at 280 nm on a Perkin–Elmer LS50B fluo-
rimeter (Perkin–Elmer Corp., Norwalk, CT, USA) at room temperature
in aqueous solution (pH 3.6). Peptide concentration was 2 mm as deter-
mined by UV absorption of the Trp residues and the AMPP chromo-
phore.
3
Periodic boundary conditions for a water box of size 40”40”30 Š con-
taining ~1500 molecules served as explicit solvent model. Experimental
NMR derived distance constraints were applied either all of the time
with full strength (50 kcalmolꢀ1 ꢀ2) or not at all. After minimization of
Š
the whole system molecular dynamics were started at 10 K for 10 ps.
Heating to 100 K proceeded with a time constant of 1 ps for the coupling
to the temperature bath during 10 ps (with NMR constraints) or 100 ps
(unrestrained). The increase in temperature to 300 K was performed
more slowly (5 ps time constant). In some cases even further heating to
500 K was applied. Individual conformations were saved each 5 ps.
IR spectroscopy: IR spectra were obtained at 258C on 1 mm peptide sam-
ples dissolved in CD3OD (Merck, Darmstadt) with an IFS66 FTIR spec-
trophotometer from Bruker (Rheinstetten, Germany) using a cuvette
with CaF2 windows (path length 220 mm). Photoisomerization from trans-
azo to cis-azo was achieved with light of 355 nm from a 1000 W HgXe
high-pressure lamp (LOT, Darmstadt) combined with UG1 and WG 320
glass filters (Schott, Mainz). For the irradiation to the trans-azo form a
Schott KL2500 LCD cold-light source together with the glass filters KG2
and GG 400 (Schott, Mainz) were used.
Results and Discussion
NMR analysis: NMR spectra of the hairpin peptide were recorded in
water, CD3OH and CD3OD at ꢀ10, 0, 20, and 308C on a Bruker DRX
500 and an AV900 spectrometer equipped with pulsed-field-gradient
(PFG) accessories and a cryoprobe in the case of the 900 MHz spectrom-
eter. Resonance assignments of the cis- and trans isomer were performed
according to the method of Wüthrich[10] and are reported in Table S1 and
S2 of the Supporting Information. The 2D TOCSY was recorded with a
spin-lock period of 70 ms using the MLEV-17 sequence for isotropic
mixing.[11] For the cis-azo isomer 34 experimental distance constraints
were extracted from 2D NOESY[12] and ROESY[13] experiments at 20
and 308C with mixing times of 150 to 200 ms (see Supporting Informa-
tion, Table S3). For the trans-azo isomer 47 experimental distance con-
straints were derived from 2D ROESY experiments at 0 and 208 with
mixing times of 100 and 150 ms (see Supporting Information, Table S4).
In the search for a suitable derivative of azobenzene our
molecular modeling suggested that a recently proposed met-
a,meta substitution of the cis azobenzene[9] might be more
suitable than our previously used AMPB,[6] to mimic the
type I’ b-turn that is required for a b-hairpin. A spacing of
the amino and carboxy function from the aromatic rings by
single methylene groups not only simplifies chemistry by de-
coupling of the respective electronic systems, but also allows
for the desired geometry. The N-Fmoc-protected photo-
switch
[3-(3-aminomethyl)phenylazo]phenylacetic
acid
For 1H diffusion measurements at 208C in CD3OH stimulated echo ex-
periments with bipolar gradients and a diffusion time of 100 ms were per-
formed in a pseudo 2D fashion by incrementing the gradient strength
from 1 Gcmꢀ1 to 60 Gcmꢀ1 in 10 steps. The gradient strength was cali-
brated to a diffusion constant of 18”10ꢀ10 m2 sꢀ1 for H2O in D2O at
300 K. Only well-resolved signals were used for extracting diffusion con-
stants from the mono-exponential signal decay.
(AMPP) was synthesized as outlined in Scheme 1 by a sim-
Structure calculation: Structure calculations and evaluations were per-
formed with the INSIGHTII 2000 software package (Accelrys, San
Diego, CA) on Silicon Graphics O2 R5000 computers (SGI, Mountain
View, CA). Hundred structures were generated from the distance-bound
matrices. Triangle-bound smoothing was used. The NOE intensities were
converted into interproton distance constraints using the following classi-
fication: very strong (vs) 1.7–2.3Š, strong (s) 2.2–2.8 Š, medium (m)
2.6–3.4 Š, weak (w) 3.0–4.0 Š, very weak (vw) 3.2–4.8 Š and the distan-
ces of pseudo atoms were corrected as described by Wüthrich.[10] The
structures were generated in four dimensions, then reduced to three di-
mensions with the EMBED algorithm and optimized with a simulated
annealing step according to the standard protocol of the DG II package
of INSIGHT II. All hundred structures were refined with a short MD-
SA protocol: After an initial minimization, 5 ps at 300 K were simulated
followed by exponential cooling to ~0 K during 10 ps. A time step of 1 fs
was used with the CVFF force field while simulating the solvent H2O
Scheme 1. Synthesis of the N-Fmoc-protected photoswitch [3-(3-amino-
methyl)-phenylazo]phenylacetic acid (AMPP). a) Fmoc-OSu, Et3N, RT;
b) NH4Cl, Zn powder, RT; then FeCl3, 08C; c) AcOH.
with a dielectric constant of 80.0. The experimental distance constraints
ꢀ2
were applied at every stage of the calculation with 50 kcalmolꢀ1
Š
.
After simulated annealing with DISCOVER the structures were sorted
according to final energies and the energy lowest were analyzed. No sig-
nificant violations of experimental constraints occurred for any of the cal-
culated cis-azo structures. However, for the trans-azo isomer distance
constraints between the methylene group of the AMPP at the amino end
and the adjacent phenyl ring had to be removed from the structure calcu-
plified procedure compared with that reported previously.[8]
Its incorporation into the peptide sequence proceeded in a
straightforward manner applying the methods reported for
other azobenzene derivatives.[14] Despite the hydrophobicity
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Hairpin Peptide
FULL PAPER
of the chromophore the peptide is soluble at millimolar con-
centrations in water and methanol. A tendency towards ag-
gregation was observed at high concentrations in aqueous
solution, but NMR diffusion and dilution studies clearly
showed that in methanol the peptide exists in a monomeric
form. In fact, the diffusion constants at 208C in CD3OH of
1.9ꢁ0.1”10ꢀ10 m2 sꢀ1 and 2.0ꢁ0.1”10ꢀ10 m2 sꢀ1 for the cis-
azo and trans-azo isomer, respectively, are independent of
concentrations between 50 mm and 2 mm and are in agree-
ment with a somewhat higher compactness for the cis-azo
than for the trans-azo molecules in line with the conforma-
tional analysis.
and shows an unfolding midpoint at 728C,[3c] substitution of
the turn residues Asn-Gly by AMPP destabilizes the hairpin
considerably. The sensitivity of b-hairpins to the conforma-
tional propensities of the turn residues is well known[1] and
was exploited in our photoresponsive peptide in order to op-
timize the influence of the chromophore on the conforma-
tion of the peptide. Comparison with the CD spectra of the
original sequence (Figure 1 in ref. [3c]) allows for an esti-
mate of the b-hairpin content of 50% at 58C in water for
the cis-azo photostationary state generated by illumination
at 360 nm. Irradiation at 430 nm reduces the folded fraction
to 18%. At 308C the CD spectrum of the trans-azo isomer
displays the typical characteristics of a b-extended confor-
mation with a pronounced positive signal below 200 nm and
a minimum close to 215 nm albeit of moderate intensity.
However, the hairpin fold is almost completely lost as indi-
cated by the very weak CD intensity at 228 nm (Figure 2,
top). Contrarily, the cis-azo isomer is still significantly
folded with a 25% hairpin content. The temperature depen-
dent CD signal of the cis-azo isomer can be fit by a sigmoid
curve, which suggests a two-state transition with a melting
temperature of 258C. Importantly, the thermal unfolding as
well as the isomerization transitions between cis- and trans-
azo configuration are both fully reversible and CD spectra
in the photostationary states before and after thermal excur-
sions or after several isomerization events are identical
within the limits of error (data not shown). Addition of
methanol led to a linear increase in CD signal intensity in
the range from 0 to 30% methanol (v/v) reaching plateau
values at about 1:1 water/methanol mixtures with a CD
spectrum (data not shown) that closely matches the CD
curve of a fully folded tryptophan zipper in shape and inten-
sity.[3] The stabilizing effect of methanol for b-hairpins has
been observed before and has been attributed to a destabili-
zation of the unfolded form.[15]
Circular dichroism: The CD spectra of the AMPP-peptide
in the predominantly cis-azo isomeric state compare well
with those reported for tryptophan zippers[3] and are, there-
fore, indicative of differentially populated b-hairpin struc-
tures (Figure 2). The characteristic maximum at 228 nm
Structural analysis of the cis-azo isomer: NMR spectroscopy
was employed for confirming the b-hairpin structure of the
AMPP-peptide in the cis-azo configuration. As b-hairpin
peptides are usually in a fast equilibrium between the folded
and unfolded form, only an average resonance position is
observed in NMR spectroscopy, which depends on the frac-
tion of folded molecules.[1,3,15] Nevertheless, characteristic
NOE values can be observed because of the strong bias of
the average NOE towards short distances and, thus, folded
forms.[16] Methanol was used as the solvent, as it is known to
prevent aggregation and to stabilize the b-hairpin structure
(see above). Extensive line broadening precluded NMR
conformational analysis at low temperatures, but at 308C
resonances of all amino acids could be observed and as-
signed for both the cis-azo and trans-azo isomer. CD meas-
urements at this temperature in 100% methanol showed a
hairpin content of 45% for the cis-azo photostationary state
while the trans-azo isomer displayed only a very weak signal
at 228 nm (data not shown). Characteristic NMR NOE
values confirmed the hairpin structure for the cis-azo isomer
and allowed calculation of an NMR structural ensemble
Figure 2. Top: CD spectra of AMPP-peptide in water (45 mm), pH 3.7, at
308C in the cis-azo (red) and trans-azo (blue) photostationary state.
Bottom: Thermal unfolding monitored by the dichroic signal at 228 nm.
The vertical line indicates the temperature that corresponds to the spec-
tra shown in the upper panel.
originates from a stacking of the tryptophan residues and
allows quantifying the folded fraction:[3] As some of the pre-
viously studied tryptophan zippers exhibit clear plateaus
above and/or below the melting points in thermal unfolding,
the CD intensity at 228 nm for a theoretically 100% folded
sample can be deduced and used as basis for further calcula-
tions of fractions folded. While the original sequence (called
“Trpzip2” in ref. [3]) is fully folded at room temperature
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C. Renner et al.
with a backbone rmsd of 1.3Š for the ten energy lowest
structures (Figure 3). While individual side chain conforma-
tions vary substantially, Figure 3clearly shows the clustering
Figure 4. Backbone superimposition of the energy lowest NMR structure
of the AMPP-peptide as cis-azo isomer (green ribbons) on the original
tryptophan zipper (violet ribbons, PDB code: 1LE1). The AMPP chro-
mophore is colored in orange and side chains of only the tryptophan resi-
dues are depicted for both peptides. The two views are rotated by 908
around a horizontal axis.
proved. No transitions between the slightly different con-
formers of the NMR ensemble were observed and each
structure remained in its original backbone conformation
with changes only in the side chain orientations. Remarka-
bly, the energy lowest NMR structure was also extremely
stable in MD simulations without the experimental con-
straints. During 900 ps of unconstrained MD at 300 K fol-
lowed by 500 ps at 500 K only minor alterations were ob-
served. As before, a more regular b-hairpin geometry was
adopted by the peptide and the corresponding hydrogen
network was observed. No indication of unfolding could be
seen during the 500 ps at 500 K.
Figure 3. Superimposition on backbone atoms of the ten energy lowest
NMR structures of the cis-azo isomer. The AMPP chromophore is col-
ored in orange; green ribbons highlight the backbone conformation; side
chains are depicted in blue for tryptophan residues and gray for all
others; the two views are rotated by 908 around a horizontal axis.
of tryptophan side chains on one side of the b-hairpin and
of the other side chains on the other side. Figure 4 depicts a
comparison of the NMR structure of the AMPP-peptide in
the cis-azo configuration with the NMR structure of the
original tryptophan zipper peptide (PDB code: 1LE1). A re-
markable agreement is observed for the antiparallel b-
strands as well as for the tryptophan side chains. In the turn
region only moderate differences between the Asn-Gly di-
peptide of the original sequence and the AMPP chromo-
phore are visible. While certainly not perfect, the novel
AMPP w-amino acid is apparently able to act as a bI’-turn
mimetic in the cis-azo isomeric form.
Molecular dynamics (MD) simulations were performed
starting from individual conformations of the NMR structur-
al ensemble to refine the structures (using the NMR derived
distance constraints) or test their stability (unrestrained
MD). In constrained simulations of 200 ps at 300 K no
changes of overall conformation were observed, as anticipat-
ed, but the local geometry of the hydrogen bonds that
would be expected for a typical b-hairpin was markedly im-
Structural analysis of the trans-azo isomer: The trans-azo
AMPP is clearly not compatible with the bI’-turn geometry
and, thus, prevents formation of a fully folded hairpin.
Again distance constraints could be derived from NMR
spectra, although the folded fraction is low at temperatures
where resonance assignment is possible. Almost all con-
straints for structure calculations where obtained from
ROESY spectra recorded at 208C. The resulting NMR
structural ensemble displayed two clearly distinct conforma-
tional families with frequencies of 75% for the first and
25% for the second (Figure 5). Both minor and major con-
formation satisfied all 47 NMR-derived distance constraints
that were used in the structure calculation. While the choice
to superimpose structures on the second peptide strand (and
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Chem. Eur. J. 2006, 12, 1114 – 1120

Hairpin Peptide
FULL PAPER
Figure 6. Comparison of trans-azo (green/orange) and cis-azo isomer
(gray).
trans-azo isomer of the AMPP-peptide exhibits a reduction
of the fluorescence intensity by almost 50% compared with
the cis-azo isomer due to the lower degree of hairpin struc-
ture and, possibly, energy transfer to the AMPP chromo-
phore. Since it is known that the tryptophan fluorescence
strongly depends on its surroundings, the pronounced
change in fluorescence intensity is a sensitive probe for cor-
rect packing of the tryptophan side chains in the b-hairpin.
Complementary to fluorescence as a means of observing
side chain interactions, IR spectroscopy can detect backbone
conformations and hydrogen bonding.
Figure 5. Superimposition on backbone atoms of the second peptide
strand (Lys6 to Lys10) of the ten energy lowest NMR structures of the
trans-azo isomer. For the major conformation the AMPP chromophore is
colored in orange; green ribbons highlight the backbone conformation;
the minor conformation is depicted in blue.
the AMPP) is somewhat arbitrary, the difference between
the two conformations can clearly be seen: Depending on
the orientation of the chromophore the first half of the pep-
tide sequence may be positioned on either side of the
second half. One has to keep in mind that the compact
structures of Figure 5 are in fast exchange with disordered
and, likely, more open structures that are not detected by
NMR.
Figure 6 depicts a comparison of the energy lowest struc-
ture of the trans-azo and the cis-azo isomer. While the two
five-residue strands are always in an extended conformation,
the twisted antiparallel alignment observed for the cis-azo
isomer transforms upon isomerization to the trans-azo state
to an irregular arrangement, that for one trans-azo confor-
mational family (depicted in Figure 6) is even close to a par-
allel orientation. All hydrogen bonds present in the b-hair-
pin are lost in the transition to the trans-azo configuration
underlining the marked differences between the conforma-
tional properties of the two isomers. It is important to re-
member that for both isomeric forms substantial structural
fluctuations do occur and that Figure 6 only displays one
representative conformation, for sake of clarity, not the full
NMR structural ensembles.
Figure 7. Tryptophan fluorescence before (trans-azo, blue curve) and
after irradiation at 360 nm (cis-azo, red curve); excitation wavelength:
280 nm.
Fourier-transform IR spectroscopy: The results from FTIR
absorption experiments are presented in Figure 8. The
upper panel shows the absorption spectrum of the AMPP-
peptide dissolved in deuterated methanol at 258C (solvent
contributions subtracted), which in the amide I band is char-
acterized by a pronounced peak at 1675 cmꢀ1 and a broad
shoulder extending to 1620 cmꢀ1. Upon irradiation at
355 nm and thus increase of the cis-azo isomer, an absorp-
tion increase at 1640 cm1 and a decrease at 1660 cmꢀ1 is ob-
served, which was fully reversed at irradiation at l >400 nm
Tryptophan fluorescence: The stability of tryptophan zippers
is mainly derived from the stacking interactions of the tryp-
tophan side chains.[3] The enhancement of fluorescence
emission that results from these interactions can be used to
monitor the degree of folding. In fact, a strong difference
between the two isomers was observed (Figure 7). The
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1119

C. Renner et al.
schungsgemeinschaft (SFB 533, grants A8 and B9 and SFB 563, grant
C4).
(Figure 8, bottom). These absorption changes reflect the
structural changes of the peptide. The increased absorption
around 1640 cmꢀ1 in the cis-azo state is in the spectral range
that is characteristic for b-sheet[17] and b-hairpin[18] absorp-
tion and confirms the interpretation of increased b-hairpin
formation when the chromophore is in the cis-azo configura-
tion. Weaker absorption changes at smaller wave numbers
are due to changes of the amide II absorption and of the
chromophore. The strong absorption changes in the amide I
range show, that ultrafast time-resolved IR spectroscopy can
be used for the direct observation of the fastest parts of the
light-induced folding and unfolding processes of the newly
developed chromopeptides.
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Figure 8. Top: FTIR-absorption spectrum of the AMPP-peptide at 258C;
bottom: Absorption difference spectra induced by irradiation at 355 nm
(trans ! cis, red curve) and at l >400 nm (cis ! trans, blue curve). The
strong absorption changes around 1650 cmꢀ1 are due to the different b-
hairpin content of the cis-azo and trans-azo isomer.
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In summary, we have designed, synthesized and character-
ized a b-hairpin peptide where the degree of folding is con-
trolled by the novel AMPP chromophore that can mimic a
type I’ b-turn in the cis-azo isomeric state. The b-hairpin
structure of the cis-azo isomer was confirmed and deter-
mined by NMR spectroscopy, while the trans-azo isomer ex-
hibits more disordered and irregular conformations. Light-
induced folding can be monitored by pronounced changes in
the CD, IR and fluorescence spectra allowing for time-re-
solved folding studies of b-structures with unprecedented
time resolution. Furthermore, ligation methods, which are
now routinely applied in biochemistry, might allow incorpo-
ration of such photosensitive b-hairpins in natural protein
structures resulting in proteins with built-in light switches.
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Acknowledgements
S.L.D. wishes to thank the Alfried Krupp von Bohlen und Halbach foun-
dation for a fellowship. The study was supported by the Deutsche For-
Received: August 13, 2005
Published online: November 18, 2005
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Chem. Eur. J. 2006, 12, 1114 – 1120