Site-directed spin labeling of a collagen mimetic peptide

Article abstract of DOI:10.1002/chem.201303290

At every turn: Electron paramagnetic resonance spectroscopy was used to investigate the dynamics of triple helix folding/unfolding of host-guest collagen mimetic peptide with a spin-labeled central triplet in Ac-(Gly-Pro-Hyp)₇-Gly-Gly-NH₂ (see figure). Copyright

Full text of DOI:10.1002/chem.201303290

COMMUNICATION
DOI: 10.1002/chem.201303290
Site-Directed Spin Labeling of a Collagen Mimetic Peptide
Jianbing Jiang,[a] Longfei Yang,[b] Qiaoying Jin,[a] Wude Ma,[a] Luis Moroder,[c] and
Shouliang Dong*[a, d]
Collagen as the most abun-
dant protein in mammals con-
sists of three chains that are
built up of repeating Xaa-Yaa-
Gly units with all peptide
bonds in trans conformation
and most often with proline in
Xaa and (4R)-hydroxyproline
in the Yaa position. This type
of sequence favors
a left-
handed poly-Pro-II helix con-
formation and the intertwining
of the three chains into the
unique right-handed triple
helix.[1] The X-ray structure
analysis of collagen mimetic
peptides (CMPs) of repeating
Xaa-Yaa-Gly triplets show Cg-
endo puckers of the Pro resi-
dues in Xaa and Cg-exo puck-
Figure 1. The relative mobility and environmental changes of spin labels with collagen folded/unfolded states.
ers of the Hyp residues in Yaa position.[2] Studies on struc-
tural factors that stabilize the triple helical fold with proline
derivatives led to the conclusion that besides the interchain
hydrogen bond between the NH group of Gly of one strand
and the CO group of Pro of the adjacent strand, stereoelec-
tronic effects that favor the all trans-peptide bond conforma-
tion and the correct puckering are decisively affecting the
structural stability.[1c,3] Recent studies by Erdman and Wen-
nemers with proline derivatives in the Yaa position that ex-
hibit preferences for the Cg-endo puckering, but a trans
amide conformer, contradict this conclusion showing that
the ring puckering is less important for the stability of colla-
gen if the trans/cis amide conformer ratio favors formation
of the triple helix.[4]
The aim of the present study was to investigate whether
electron spin resonance spectroscopy (ESR) could yield new
information on the dynamics of triple helix folding/unfolding
as a useful alternative to the other spectroscopic techniques
applied so far. Indeed this type of spectroscopy has become
popular for studying conformational changes and unfolding
processes of proteins[4] as many ESR spectral parameters,
such as peak-to-peak height, peak-to-peak width and rota-
tional correlation time can accurately reflect relative mobili-
ty and environmental changes of spin labels (Figure 1).[5]
For the design and synthesis of a suitable spin-labeled
CMP, the results of a recent detailed study on the equilibri-
um constants of acetyl-(4R)-Pro(X)-OMe (X=ammonium
or acetylamide) by NMR spectroscopy[6] were taken into ac-
count. Although for the ammonium derivative a significantly
reduced preference for the trans conformation was ob-
served, a trans conformer preference similar to that of (4R)-
hydroxyproline was recovered upon acetylation of the g-
amino group. Correspondingly, Ac-(Gly-Pro-Hyp)7-Gly-Gly-
[a] J. Jiang,+ Q. Jin, W. Ma, Prof. Dr. S. Dong
School of Life Sciences, Lanzhou University
222 Tianshui South Road, Lanzhou 730000 (China)
E-mail: dongsl@lzu.edu.cn
[b] L. Yang+
Department of Medical Laboratory and Research Center
Tangdu Hospital, Fourth Military Medical University
Xi’an 710038 (China)
[c] Prof. Dr. L. Moroder
Bioorganic Chemistry, Max Planck Institute of Biochemistry
Am Klopferspitz 18, 82152 Martinsried (Germany)
[d] Prof. Dr. S. Dong
Key Laboratory of Preclinical Study for New Drugs of
Gansu Province, Lanzhou University, 222 Tianshui South Road
Lanzhou 730000 (China)
[+] These authors contributed equally to this work.
[7]
NH2 was selected as the CMP in which the Hyp residue of
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201303290.
the central triplet was replaced by (2S,4R)-aminoproline
Chem. Eur. J. 2013, 19, 17679 – 17682
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17679

(Amp) to allow for insertion of an nitroxide radical by side-
chain acylation of the related Amp containing host–guest
CMP with a suitable spin-label derivative.
Among the nitroxide spin probes frequently utilized for
site-specific labeling of peptides and proteins, such as
tive band near 225 nm and a negative maximum around
198–200 nm showed the typical pattern of poly-Pro-II heli-
ces (Figure 2A). The CD spectra shows that CMP and SLP
actually both fold into the typical collagen triple helix, but
the dichroic intensity of the negative maximum was found
to be lower for the SLP than for the Amp-peptide. Thus, the
CD signature of SLP would indicate a lower content of
triple helix than for the Amp-CMP. To test the influence of
2,2,5,5-tetramethyl-1-oxyl-3-methylmethane
thiosulfonate
(MSTL),[8] 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-car-
boxylic acid (TOAC),[9] 2,2,6,6-tetra-methylpiperidine-N-
oxyl (TEMPO),[10] and 3-carboxy-2,2,5,5-tetramethylpyrroli-
dine-1-yloxy free radical (3-carboxy-PROXYL)[11] we have
selected the latter reagent as it is readily prepared from the
commercially available 2,2,6,6-tetramethyl-4-piperidine by
known procedures.[12]
The synthesis of the Amp-CMP (2S,4R)-Fmoc-Amp-
ACHTUNGTRENNUNG
Scheme 1. Synthesis of (2S,4R)-Fmoc-AmpACTHNUTRGNEUNG(Boc)-OH: a) i) 2N NaOH
(4.0 equiv), 08C; ii) ZCl (2.5 equiv), RT, 4 h; iii) 6N HCl, pH 1, 84.7%;
b) BnBr (1.5 equiv), TEA (10 equiv), RT, 22 h, 92.8%; c) Boc2O
(2.0 equiv), DMAP (0.1 equiv), THF, RT, 17 h, 92.0%; d) 10% Pd/C, 9:1
EtOH/H2O (v/v), H2, RT, overnight, 83.0%; e) Fmoc-OSu (1.5 equiv),
2:1 THF/H2O (v/v), NaHCO3 (2.5 equiv), RT, overnight, 78.7%.
of the g-amino and a-amino groups of Amp to yield 2, the
carboxy group was protected as benzyl ester 3. Subsequent
double acylation of the g-amino group of 3 with (Boc)2O by
DMAP catalysis led to compound 4. Hydrogenation of 4
over Pd/C catalyst for removal of the Z and benzyl protect-
ing groups produced intermediate 5 for reaction with Fmoc-
OSu to afford the target derivative 6. It was obtained in an
overall yield of 47%, which is about 30% higher than that
of the previously reported synthesis.[13]
The suitably protected Amp derivative was then applied
for the synthesis of Ac-(Gly-Pro-Hyp)3-Gly-Pro-Amp-(Gly-
Pro-Hyp)3-Gly-Gly-NH2 by standard Fmoc/tBu synthesis on
solid support. As shown in Scheme S1 in the Supporting In-
formation, the resulting Amp-containing host–guest CMP
(8) was acylated in solution with an excess of 3-carboxy-
PROXYL (7) in the presence of HBTU/HOBt/DIEA
(1:1:2). Both the Amp-CMP (8) and the spin-labeled colla-
gen peptide (SLP) 9 were obtained as highly homogeneous
and well-characterized compounds.
Based on previous detailed studies on kinetics of folding/
unfolding of CMPs into the triple helical structure[14] aque-
ous solutions of peptides 8 and 9 at 1 mm concentration
were incubated at 48C for 24 h. The CD spectra with a posi-
Figure 2. CD spectra of CMP (8) and SLP (9). A) CD spectra of 8 (solid
line) and 9 (dashed line) at 48C; B) CD melting curves of 8 (solid line)
and 9 (dashed line) triple helices; C) the first derivative of melting curves
of 8 and 9 triple helices versus temperature.
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Spin Labeling of a Collagen Mimetic Peptide
COMMUNICATION
Amp and its spin-labeled derivative on the stability of the
triple helix, the thermal denaturation was monitored by
changes in dichroic intensities at 225 nm in the temperature
range from 5 to 708C. As shown in Figure 2B both peptides
display a cooperative transition with very similar midpoints
Tm of 32.8 and 32.28C for 8 and 9, respectively, as deter-
mined by the derivative of the melting curves (Figure 2C).
Compared to the parent CMP, that is, Ac-(Gly-Pro-Hyp)7-
Gly-Gly-NH2, for which a Tm of 438C was reported under
identical conditions,[7] the stability of the two collagen pep-
tides 8 and 9 is reduced by 108C. For the Amp-peptide as
trifluoroacetate salt, such weaker stability was predictable
on the basis of the lower trans conformer preference as in-
duced by the g-ammonium group,[6] whereas the unprotonat-
ed Amp, under basic conditions, was reported to significant-
ly stabilize the triple helix.[15] The reduced triple helix stabil-
ity of the spin-labeled peptide was unexpected as acylation
of the g-amino group was predicted to restore, to a large
extent, the preference for the trans-conformation.[6] Such
benefit may well be annulled by sterical clashes of the nitro-
xide label that lead to a stronger breathing of the triple
helix in its central part. More information on this aspect can
possibly be expected from ESR measurements.
The results from ESR spectroscopy experiments are pre-
sented in Figure 3A and Figure S9 in the Supporting Infor-
mation as the first-derivative lines of original ESR absorp-
tion spectra. They all show three-line ESR spectra referred
to as center-, low- and high-field lines, for the hyperfine in-
teraction of unpaired electron spins in water. Indeed, the
aqueous solution of nitroxide shows a typically three-line
ESR spectrum of approximately equal heights reflecting its
fast motion.[5] Varying degrees of broadening of the ESR
spectra are observed in Figures S9 and S10 in the Supporting
Information, which indicate the influence of the peptide
backbone on spin-probe mobility, consistent with experien-
ces with other nitroxide-labeled peptides.[9,16] It is well
known that ESR spectroscopy is highly sensitive to the local
structure and environmental changes around the spin label
in proteins. Upon heating the solution of peptide 9 from 5
to 708C, the ESR spectrum shows strong changes with
a broad signal, corresponding to the transition from folded
to unfolded state of the triple helix as shown by CD spec-
troscopy. The unfolding process of the triple helix results in
an increased rotational tumbling of the nitroxide probe,
which is consistent with a reduced immobilization as expect-
ed from an increased fluctuation of the spin label in unfold-
ed single chains. Similar effects of reduced constraints upon
unfolding were reported for other spin-labeled peptides and
structured proteins.[4b] In addition, the spin–spin interaction
in the triple helix, which depends on distance between the
labels, may also contribute to the broader lines of ESR spec-
trum.[17] Therefore, interspin distance measurements using
ESR technique could well add further information on trirad-
ical interactions in the triple helix, and thus allow the dy-
namics of local unfolding and folding to be monitored.
Figure 3. A) ESR spectra of SLP (9) triple helix at different temperatures
in three-dimensional view. B) The relationship between DHpp(0)/tc and
temperatures of SLP (9). Dotted line: rotational correlation times (tc) at
different temperatures; solid line: the peak-to-peak width (DHpp(0)) of
the center line at different temperatures. C) The first derivative of rota-
tional correlation time versus temperatures (solid line); normalized tc
values versus temperatures (dotted line).
and mobility. According to the ESR spectra in Figure 3A,
we could confirm the dynamics of the spin-labeled collagen
mimetic peptide as a fast motional model. Thus, the motion
of peptide can be reflected by the rotational correlation
time, tc, which is the time necessary for the spin label to
rotate through an angle of one radian given by Equa-
tion (1).[18] It accurately reflects relative motilities and
changes in the spin label motion.
The ESR spectra show a combined spectroscopy of three
spin labels for they share the same interaction environment
Chem. Eur. J. 2013, 19, 17679 – 17682
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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17681

S. Dong et al.
sffiffiffiffiffiffiffiffiffiffiffiffiffiffi sffiffiffiffiffiffiffiffiffiffiffiffiffiffi
(
)
pffiffiffi
Experimental Section
3
4
Að0Þ
Að0Þ
tc ¼ 1:19 ꢀ 10ꢁ9
ꢀ
þ
ꢁ 2 ꢀ DHppð0Þ
Aðþ1Þ
Aðꢁ1Þ
All the experimental procedures and are reported in the Supporting In-
formation.
ð1Þ
Calculated values of the peak-to-peak width of the center
line (DHpp(0)) and the correlation time (tc) by varying the
temperatures are shown in Table S1 in the Supporting Infor-
mation. The sensitivity of the ESR spectrum to the spin-
label mobility is influenced by both the rate and amplitude
of the motion. Consequently, a clear correlation between
ESR spectral shape and conformational status of the triple
helix could be established. In Figure 3, DHpp(0) and tc are
shown with increasing temperatures. The changes of these
values reflect the thermal transition from triple helix to the
unfolded state, which are consistent with the CD spectra.
From 5 to 408C, the tc values decrease almost linearly from
1.21865 to 0.19669 ns and DHpp(0) values also show a linear
decrease from 0.20935 to 0.12818 mT, proving the unfolding
process of the triple helix and the related weakening of the
restricted local structure and spin–spin interactions. The re-
lationship between tc and temperature can be viewed as
ESR melting transition of the triple helix of peptide 9. For
comparative analysis of thermal transitions monitored by
CD and ESR very slow heating rates (128Chꢁ1) were ap-
plied as required for almost equilibrium transitions of CMPs
even at high peptide concentrations.[14b,19]
Acknowledgements
The authors want to thank Prof. Claudio Toniolo (University of Padova,
Italy) for the kind advice in the ESR experiments. This work was in part
supported by the grant from National Natural Science Foundation of
China (No. 30870526).
Keywords:
(2S,4R)-aminoproline
·
collagen
·
ESR
spectroscopy · proteins · site-directed spin labeling
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The results obtained with the spin-labeled CMP fully con-
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dynamics.
Received: August 22, 2013
Published online: November 22, 2013
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Chem. Eur. J. 2013, 19, 17679 – 17682