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Kinetics and Equilibria of Cis/Trans Isomerization of Secondary Amide Peptide Bonds in

Linear and Cyclic Peptides


Khanh Nguyen, Margret Iskandar, and Dallas L. Rabenstein*
Department of Chemistry, UniVersity of California, RiVerside, California 92521
ReceiVed: January 2, 2010
The secondary amide peptide bonds that comprise up to one-third of the bonds of peptide or protein backbones
can exist as cis and trans isomers, with the trans isomer being highly favored. However, there is little quantitative
data on the kinetics and equilibria of cis-trans isomerization of secondary amide peptide bonds due to the
difculty of detecting the very small population of cis isomers. Knowledge of factors that inuence the kinetics
and equilibria of cis-trans isomerization of secondary amide peptide bonds will contribute to a more complete
understanding of the structural and dynamic behavior of the backbones of peptides and unfolded proteins and
of complex protein folding kinetics. We have characterized the kinetics and equilibria of cis-trans isomerization
of the Xaa-Yaa secondary amide peptide bonds of the linear dithiol and cyclic disulde forms of the peptides
Ac-Cys-Xaa-Yaa-Cys-His-NH
2
, where Xaa-Yaa is Ala-Phe, Phe-Ala, Ala-Tyr, and Tyr-Ala, by
1
H NMR.
Resolved resonances were observed for the Ala-methyl protons of the trans and the much less abundant cis
isomers due to differential shielding of the Ala-methyl protons of the trans and cis isomers by ring current
effects from the Phe and Tyr side chains. The population of the cis isomers was determined from the relative
intensities of the Ala-methyl resonances for the trans and cis isomers, and rate constants for cis-to-trans and
trans-to-cis isomerization were determined by the magnetization transfer NMR method. The population of
the cis isomers ranges from 0.07 to 0.12%, and the rate constants indicate that, when there is a trans-to-cis
interchange, it is rapidly followed by a cis-to-trans interchange back to the more stable trans conformation.
Although cyclization by disulde bond formation imposes conformational constraints on the peptide backbones,
cyclization is found to have relatively small affects on the dynamics of cis-trans isomerization.
Introduction
Due to its partial double bond character, the peptide bond is
rigid and planar and exists in trans and cis conformations. Even
though up to one-third of the bonds along a peptide or protein
backbone are secondary amide peptide bonds, i.e., amide peptide
bonds to nitrogen of the nonproline amino acids, there is almost
no quantitative data on the population of the cis isomer or the
kinetics of cis-trans isomerization of secondary amide peptide
bonds.
1-3
It is well established that secondary amide peptide
bonds favor the trans conformation due to less steric interaction
between side chains of anking amino acids,
4-6
and that, in the
native, functional state of folded proteins, secondary amide
peptide bonds generally exist exclusively in the trans conforma-
tion.
5-10
However, the secondary amide peptide bonds in
peptides and unfolded proteins exist in a state of dynamic
equilibrium between trans and cis conformations, with the trans
conformation much more highly populated.
Characterization of the kinetics and equilibria of cis-trans
isomerization of secondary amide peptide bonds in peptides and
unfolded proteins has been hampered by the lack of experimental
methods for detecting the very small populations of cis isomers.
Consequently, only a few studies of cis-trans isomerization of
secondary amide peptide bonds have been reported and these
have been limited to small peptides, mainly dipeptides.
1-3
Rate
constants were determined for cis-trans isomerization of the
secondary amide peptide bonds of Gly-Gly, Gly-Ala, and Ala-
Gly by a UV/visible spectral method by exploiting differences
in the absorption properties of the cis and trans conformations,
and the dependence of the cis population on the protonation
state of the dipeptides.
1
Isomerization rate constants were
determined by following spectral changes after pH jumps by
stopped ow methods. Rate constants for cis-trans isomeriza-
tion of Gly-Gly have also been measured by a UV resonance
Raman method in which photon absorption was used to
isomerize the trans isomer to the cis isomer.
2
However, the rate
constants obtained are much larger than those measured for Gly-
Gly by the UV/visible method.
1
Rate constants and thermody-
namic parameters were determined by
1
H NMR for the
secondary amide peptide bonds linking alanine to the amino
and carboxyl groups of phenylalanine and tyrosine in dipeptides
and several small peptides.
3
Conformer-specic chemical shifts
were observed for Ala-methyl groups anking the aromatic
amino acids.
Knowledge of factors that govern the kinetics and equilibria
of cis-trans isomerization of secondary amide peptide bonds
will contribute to a more complete understanding of the
structural and dynamic behavior of the backbones of peptides
and unfolded proteins and of complex protein folding kinetics.
For example, even though the cis population of each secondary
amide peptide bond in an unfolded protein is statistically very
* To whom correspondence should be addressed. E-mail:
dallas.rabenstein@ucr.edu.
J. Phys. Chem. B 2010, 114, 33873392 3387
10.1021/jp1000286 2010 American Chemical Society
Published on Web 02/05/2010
small, the necessary cis-to-trans isomerization of these peptide
bonds during the folding of a protein with all trans secondary
amide peptide bonds in the native state can give rise to complex
folding kinetics, with the rate limiting step being cis-to-trans
isomerization for a very small fraction of the protein.
11
For
example, 5% of the molecules in a proline-free variant of the
74-amino acid protein Tendamistat fold slowly, with a rate
constant of 2.5 s
-1
, due to slow cis-to-trans isomerization of
cis secondary amide peptide bonds.
11
Likewise, trans-to-cis
isomerization of a peptide bond during the folding of a protein
with a cis secondary amide peptide bond in the native state can
be the rate limiting step.
12,13
In this paper, we report the results of
1
H NMR studies of the
kinetics and equilibria of cis-trans isomerization of the Xaa-
Yaa secondary amide peptide bond in a series of peptides of
the sequence Ac-Cys-Xaa-Yaa-Cys-His-NH
2
, where Xaa-Yaa
1
is Ala-Tyr, Tyr-Ala, Ala-Phe, and Phe-Ala. Rate and equilibrium
constants for cis-trans isomerization were determined for each
peptide in both its linear dithiol and cyclic disulde forms to
determine the effect of conformational constraints imposed by
cyclization of the peptide on cis-trans isomerization of the
secondary amide peptide bonds. The rate and equilibrium
constants are among the very few reported for cis-trans
isomerization of secondary amide peptide bonds in linear
peptides
1-3
and are the rst to be reported for secondary amide
peptide bonds in cyclic peptides. The peptides were chosen for
study, as they are models for the active site of oxido-reductase
enzymes that have the Cys-Xaa-Yaa-Cys active site motif. It
has been proposed, on the basis of NMR structures, that
functional differences between the oxidized and reduced forms
of E. coli thioredoxin are related to differences in conformational
exibility in and near the active site loop of the oxidized form.
14
Experimental Section
Materials. Triuoroacetic acid (TFA) and 9-uorenyl-
methoxycarbonyl (Fmoc)-protected amino acids were purchased
from Chem-Impex International Inc. N,N-dicyclohexylcarbo-
diimide (DCC), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylu-
ronium hexauorophosphate (HBTU), and N-methyl-2-pyrroli-
done (NMP) were obtained from Applied Biosystems. Sigma-
Aldrich supplied triisopropylsilane (TIPS), R-cyano-4-hydroxy-
cinnamicacid(CHCA), piperidine(PIP), sodium3-(trimethylsilyl)-
propionate-2,2,3,3-d
4
(TMSP), and N,N-diisopropylcarbodiimide
(DIPCDI), and N,N-dimethylformide (DMF), phenol, methanol,
acetonitrile, acetic anhydride, and methyl t-butyl ether (MTBE)
were obtained from Fisher Scientic. DTT (1,4-dithiol-DL-
threitol) was obtained from Fluka. Cambridge Isotope Labora-
tories supplied NaOD (40%), DCl (35%), D
2
O, and deuterated
(98%) dithiothreitol, and Rink amide 4-methylbenzhydrylamine
(MBHA) resin (0.56 mmol/g) was purchased from NovaBiochem.
Peptide Synthesis and Purication. Peptides 1a-4a in
Table 1 were synthesized on an Applied Biosystems ABI-433A
solid phase peptide synthesizer using standard Fmoc peptide
synthesis methodology. Fmoc-MBHA with a loading capacity
of 0.56 mmol/g and a 10 times excess of each Fmoc-protected
amino acid were used for the peptide synthesis. Support-bound
peptides were cleaved from the resin and side chains deprotected
with a cleavage cocktail comprised of 88% TFA, 4.2% H
2
O,
5.8% phenol, and 2% triisopropylsilane (TIPS) by volume.
15,16
Cyclic disulde-bridged peptides (peptides 1b-4b in Table
1) were prepared by oxidation of peptides 1a-4a with trans-
[Pt(en)
2
Cl
2
]
2+
, where en is ethylenediamine, an oxidizing agent
for rapid and quantitative formation of intramolecular disulde
bonds with high selectivity.
17
The crude peptides were puried on a Varian HPLC using a
Vydac C
18
semiprep reversed phase column (10 250 mm).
Elution from the column was monitored at a wavelength of 215
nm. Peptides were eluted fromthe column with a gradient of mobile
phase A (0.1% TFA in H
2
O) and mobile phase B (0.1% TFA in
acetonitrile). Optimum retention time and resolution were achieved
with a linear gradient of 2-30% mobile phase B in 30 min.
Identities of crude peptides after cleavage from the resin and of
pure peptides isolated by reversed-phase HPLC were conrmed
by matrix-assisted, laser desorption/ionization mass spectrometry.
NMR Samples. 320 L solutions of 20 mM peptide were
prepared in 90% H
2
O/10% D
2
O at pH 3.0. TMSP was added
for a chemical shift reference. To remove any disulde peptide
formed by air oxidation in solutions of the dithiol peptides, a
3-fold excess of deuterated DTT was added, the pH was adjusted
to 7.5 with 0.1 M DCl and 0.1 M NaOD, the peptide solutions
were allowed to react with DTT for 1 h, and the pH was then
adjusted to 3.0. Sample solutions were degassed with N
2
for
30 m. NMR experiments were run immediately after degassing
the sample to minimize air oxidation.
NMR Spectroscopy. One- and two-dimensional
1
H NMR
spectra were measured at 500 MHz with a Varian Unity-Inova
spectrometer. Chemical shifts are reported relative to the methyl
resonance of TMSP at 0.000 ppm. The residual HOD resonance
was suppressed with a presaturation pulse. Two-dimensional
total correlation spectroscopy (TOCSY) and rotating frame
Overhauser effect spectroscopy (ROESY) spectra and band-
selective, homonuclear-decoupled (BASHD)-TOCSY and
BASHD-ROESY spectra were measured with standard pulse
sequences.
18-21
TOCSY and ROESY spectra were measured
with the following parameters: spectral width of 5500 Hz in
both dimensions, 8196 data points in the directly detected (F2)
TABLE 1: Peptides Studied in This Research and Chemical Shift Data for the Ala-Methyl Resonances of cis and trans Isomers
of the Indicated Secondary Amide Peptide Bonds
Ala-CH
3
chemical shift
a
peptide amino acid sequence peptide bond trans cis
1a Ac-C-A-Y-C-H-NH
2
A-Y 1.295 0.841
1b Ac-C-A-Y-C-H-NH
2
A-Y 1.280 0.922
2a Ac-C-Y-A-C-H-NH
2
Y-A 1.321 0.984
2b Ac-C-Y-A-C-H-NH
2
Y-A 1.296 1.083
3a Ac-C-A-F-C-H-NH
2
A-F 1.274 0.791
3b Ac-C-A-F-C-H-NH
2
A-F 1.211 0.886
4a Ac-C-F-A-C-H-NH
2
F-A 1.328 0.986
4b Ac-C-F-A-C-H-NH
2
F-A 1.305 1.085
a
ppm vs TMSP.
3388 J. Phys. Chem. B, Vol. 114, No. 9, 2010 Nguyen et al.
dimension, 64 transients, and 128 t1 increments. Mixing times
of 120 and 200 ms were used for TOCSY and ROESY
experiments, respectively. Shifted sine bell and Gausian apodiza-
tion were applied in the F1 and F2 dimensions, respectively.
BASHD-TOCSY and BASHD-ROESY spectra were measured
with the same parameters, with the exception that the spectral
width in the F1 dimension was less, as needed to cover a specic
band of resonances.
20,21
Rate constants for cis-trans isomerization were determined
by the inversion-magnetization transfer method using the Ala-
methyl resonances. The trans resonance of a given cis/trans pair
of resonances was selectively inverted with the pulse sequence:
23
90
x
--90
x
-t-90
(x,(y
-acquisition, where is a xed delay
time which equals 1/(2|
t
-
c
|),
t
-
c
is the chemical shift
difference in Hz of the resonances for the trans and cis isomers,
and t is a variable delay during which magnetization transfer
takes place by interchange between the cis and trans isomers.
22
The more intense trans resonance was selectively inverted, and
the transfer of inversion to the less intense cis resonance was
monitored as a function of t. Typically, inversion-magnetization
transfer spectra were measured at 14-16 t values ranging from
0.0001 s to at least 5 times the longest T
1
of the cis and trans
resonances; T
1
values were determined in a separate experiment
by the inversion-recovery method. For each t value, 256
transients were collected.
Results
Assignment of
1
H NMR Spectra. The
1
H NMR spectrum
of each peptide in Table 1 is comprised of resonances for the
peptide with all peptide bonds in the trans conformation, and a
much less intense resonance for the Ala-methyl protons of the
isomer in which the Ala-Phe, Phe-Ala, Ala-Tyr, or Tyr-Ala
peptide bond is in the cis conformation. To illustrate, the Ala-
methyl region of the spectrum of peptide 2b is shown in Figure
1. Only the Ala-
12
CH
3
resonance of the isomer having the trans
conformation across the Tyr-Ala peptide bond is observed in
spectrum A. The Ala-
12
CH
3
resonance of the cis isomer and
the
13
C-satellites of the Ala-
12
CH
3
resonance of the trans isomer
are also observed in spectrum B, which is plotted with a 320
times vertical scale expansion.
The resonances of the all-trans peptide were assigned using
two-dimensional BASHD-TOCSY and BASHD-ROESY spec-
tra. First, the backbone amide NH resonances were assigned to
specic amino acids in the peptide by measuring BASHD-
TOCSY spectra with F1-band selection of the amide NH region.
The trans conformation across each secondary amide peptide
bond was established by dipolar cross peaks in BASHD-ROESY
spectra measured with F1-band selection of the amide NH
region. To illustrate, the NH (F1)-C
R
H (F2) region of the
BASHD-ROESY spectrum of peptide 2b is shown in Figure 2,
with the C
R
H
i
-NH
i+1
cross peaks that establish the trans
conformation across the secondary amide peptide bonds identi-
ed. The inset shows the overlapped TyrNH-TyrC
R
H and
Figure 1. The Ala-methyl region of the 1D
1
H NMR spectrum of a 22 mM solution of the disulde form of Ac-Cys-Tyr-Ala-Cys-His-NH
2
(peptide 2b) in 90% H
2
O/10% D
2
O at pH 2.91 and 25 C. The spectrum was measured by the single pulse method, with suppression of the H
2
O
resonance by presaturation. Spectrum B is plotted with a vertical scale 320 that of spectrum A.
Figure 2. The NH(F
1
)-C
R
H(F
2
) region of the BASHD-ROESY
spectrum of 22 mM peptide 2b in 90% H
2
O/10% D
2
O at pH 2.91 and
25 C, measured with F
1
-band selection of the NH region. The
NH
i
-C
R
H
i+1
cross peaks that establish the all-trans conformation for
the most abundant isomer are identied. The cross peak in the inset is
from the standard ROESY spectrum; it is comprised of two overlapping
cross peaks (TyrNH-TyrC
R
H and TyrC
R
H-AlaNH) that are resolved
in the BASHD-ROESY spectrum as a result of collapse of multiplets
to singlets in the F
1
dimension.
Isomerization of Secondary Amide Peptide Bonds J. Phys. Chem. B, Vol. 114, No. 9, 2010 3389
TyrC
R
H-AlaNH cross peaks in the regular ROESY spectrum,
to demonstrate the signicant increase in resolution achieved
by collapse of multiplets to singlets in the F1 dimension in the
BASHD-ROESY spectrum.
21
The Ala-methyl resonance at 1.083 ppm in Figure 1 was
assigned to the isomer having the cis conformation across the
Tyr-Ala peptide bond by one-dimensional magnetization transfer
experiments. The Ala-methyl resonance at 1.296 ppm for the
trans isomer was selectively inverted, and the intensity of the
cis resonance measured as a function of the exchange time in
the inversion-magnetization transfer pulse sequence. As de-
scribed below, the intensity of the cis resonance decreased as
the exchange time was increased, indicating the Ala-methyl
resonance assigned to the trans isomer is linked by chemical
exchange to the resonance at 1.083 ppm. The chemical shifts
of the Ala-methyl protons of the trans and cis isomers are listed
in Table 1. The Ala-methyl resonances assigned to the cis
isomers are shifted upeld of the trans resonances, with the
magnitude of the shift being larger for the Ala-Phe and Ala-
Tyr sequence isomers.
3
Equilibrium Constants for Cis/Trans Isomerization. Equi-
librium constants for cis/trans isomerization (K
eq
) [trans]/[cis])
of the Tyr-Ala, Phe-Ala, Ala-Tyr, and Ala-Phe peptide bonds
of the peptides listed in Table 1 were determined using the
relative areas of the Ala-methyl cis and trans resonances.
Because of the large differences in intensity of the cis and trans
resonances (Figure 1), relative resonance intensities were
determined using the upeld
13
C-satellite resonance of the Ala-
methyl trans resonance, which comprises 0.554% of the trans
resonance intensity. Due to the very low intensities of the
13
C-
satellite and cis resonances, relative areas were determined from
the relative masses of the
13
C-satellite and cis resonances. The
percent cis isomer, equilibrium constants for cis/trans isomer-
ization, and the difference in free energy of the cis and trans
isomers for each peptide are reported in Table 2.
Kinetics of Cis/Trans Isomerization. Rate constants were
determined by selective inversion of the Ala-methyl resonance
for the trans isomer of each cis/trans pair. The intensity of the
cis resonance was then measured as a function of the length of
the exchange delay in the inversion-magnetization transfer pulse
sequence. To illustrate, inversion-magnetization transfer spectra
for peptide 2b are shown in Figure 3. The decrease in intensity
of the cis resonance as the delay time is increased is due to
transfer of inverted trans resonance intensity to the cis resonance
by trans-to-cis interchange. At longer delay times, the cis
resonance intensity recovers to its equilibrium intensity by T
1
relaxation.
Rate constants for cis-to-trans isomerization (k
ct
) for peptides
1-4 in Table 1 were determined from the dependence of the
intensity of the cis resonance on delay time by a nonlinear least-
squares analysis.
23
To illustrate, the nonlinear least-squares t
of the inversion-magnetization transfer spectra plotted in Figure
3 is presented in Figure 4; a value of 1.3 (0.08 s
-1
was obtained
for rate constant k
ct
. The rate constant for trans-to-cis isomer-
ization (k
tc
) at 25 C was then calculated from K
eq
and k
ct
(k
tc
) k
ct
/K
eq
).
Rate constants for cis-to-trans and trans-to-cis isomerization
for all of the peptides listed in Table 1 are summarized in Table
2. Rate constants for peptides 1a-4a and 2b were measured
directly at 25 C. Rate constants for peptides 1b, 3b, and 4b
were measured at elevated temperatures; rate constants at 25
C were then calculated using activation parameters obtained
with the Eyring equation.
Discussion
Even though up to one-third of the bonds that form peptide
and protein backbones are secondary amide peptide bonds, there
is a paucity of data on the kinetics and thermodynamics of their
cis-trans isomerization.
1-3
The spectra in Figure 1 and the
results in Table 2 demonstrate why this is the case. The
resonance for the Ala-CH
3
protons of the cis isomer is much
TABLE 2: Population of the cis Conformation, Equilibrium Constants, and Rate Constants for cis/trans Isomerization of the
Indicated Secondary Amide Peptide Bonds, and Free Energy Differences of the cis and trans Isomers of the Peptides in Table
1
a
peptide peptide bond % cis K
eq
G (kcal/mol) k
ct
(s
-1
) k
tc
(s
-1
)
1a A-Y 0.081 ( 0.0003 1232 ( 4 4.22 2.0 ( 0.09 (1.6 ( 0.07) 10
-3
1b A-Y 0.074 ( 0.0003 1327 ( 5 4.26 0.60 ( 0.03 (0.45 ( 0.02) 10
-3
2a Y-A 0.070 ( 0.0004 1422 ( 9 4.30 1.4 ( 0.05 (0.99( 0.04) 10
-3
2b Y-A 0.097 ( 0.0008 1027 ( 9 4.11 1.3 ( 0.08 (1.2 ( 0.08) 10
-3
3a A-F 0.098 ( 0.0007 1019 ( 7 4.43 0.86 ( 0.06 (0.85 ( 0.06) 10
-3
3b A-F 0.087 ( 0.002 1082 ( 16 4.14 0.37 ( 0.05 (0.34 ( 0.05) 10
-3
4a F-A 0.074 ( 0.0003 1353 ( 6 4.27 1.2 ( 0.08 (0.91 ( 0.06) 10
-3
4b F-A 0.12 ( 0.001 843 ( 10 3.99 1.1 ( 0.1 (1.3 ( 0.2) 10
-3
a
25 C.
Figure 3. Resonances for the Ala-methyl protons of the isomers of
peptide 2b having the cis and trans conformations across the Tyr-Ala
secondary amide peptide bond as a function of the length of the
exchange time t in the inversion-magnetization transfer experiment.
The sample was 22 mM peptide 2b in 90% H
2
O/10% D
2
O at pH 2.91
and 25 C. The trans resonance was selectively inverted. The resonances
for the cis isomer are plotted with a vertical scale expansion 1000
that of the resonances for the trans isomer. The exchange times were
0.0001, 0.0100, 0.1000, 0.2500, 0.4000, 0.6000, 0.8000, 1.0000, 1.5000,
2.0000, 3.0000, 4.0000, 5.0000, and 6.0000 s.
3390 J. Phys. Chem. B, Vol. 114, No. 9, 2010 Nguyen et al.
less intense than the Ala-
12
CH
3
resonance for the trans isomer
(Ala-
12
CH
3
trans:cis ) 1016:1), and is even signicantly less
intense than the
13
C-satellite resonances for the trans isomer,
which each comprise 0.554% of the total intensity of the Ala-
CH
3
resonance for the trans isomer. Furthermore, the very weak
Ala-CH
3
resonance for the cis isomer of peptide 2b is observable
only because of differential shielding of the Ala-methyl cis and
trans resonances by a ring current effect from the aromatic ring
of the adjacent Tyr residue.
3
The results reported in Table 2 indicate that the cis isomer
of the Tyr-Ala, Phe-Ala, Ala-Tyr, and Ala-Phe peptide bonds
of the linear and cyclic peptides in Table 1 is present at very
low abundance, ranging from 0.070 to 0.12%, and that cycliza-
tion has relatively little effect on the population of the cis isomer.
The results indicate that constraints imposed on the peptide
backbone by cyclization also have relatively little effect on the
dynamics of cis-trans isomerization: rates for both cis-to-trans
and trans-to-cis interchange of the secondary amide peptide
bonds of the Tyr-Ala and Phe-Ala sequence isomers are
essentially the same for the linear and cyclic forms of peptides
2 and 4, while those for the Ala-Tyr and Ala-Phe peptide bonds
of the cyclic forms of peptides 1 and 3 are decreased by only
a factor of 2-3.
For both the linear and cyclic forms of the peptides, the rates
of cis-to-trans isomerization of the Phe-Ala, Ala-Phe, Tyr-Ala,
and Tyr-Ala peptide bonds are approximately a 1000-fold faster
than the rates of trans-to-cis isomerization. Thus, while cis-trans
isomerization of secondary amide peptide bonds causes con-
formational heterogeneity along the backbone of peptides and
unfolded proteins, the vast majority of each secondary amide
peptide bond is in the trans conformation, and when there is a
trans-to-cis interchange, it is quickly followed by a cis-to-trans
interchange to the much more stable trans conformation. For
example, the trans isomer of the Ala-Tyr peptide bond of peptide
1a has a t
1/2
value of 433 s as compared to 0.347 s for the cis
isomer. The trans isomer of the Ala-Tyr peptide bond of 1b is
even kinetically more stable, with a t
1/2
value of 1540 s, while
t
1/2
of the cis isomer is 1.16 s.
Nevertheless, the relatively slow rate (on the protein folding
time scale) of cis-to-trans interchange can cause complex protein
folding kinetics. As mentioned above, 5% of the molecules in
a proline-free variant of the 74 amino acid protein Tendamistat
fold slowly, with a rate constant of 2.5 s
-1
, due to slow cis-to-
trans isomerization of the secondary amide peptide bonds in
the cis conformation.
11
If the secondary amide peptide bonds
in the cis conformation are uniformily distributed along the
backbone of the unfolded protein, a slow folding rate for 5%
of the unfolded protein corresponds to 0.07% of each secondary
amide bond in the cis conformation, in qualitative agreement
with the populations found for the cis isomers of the secondary
amide peptide bonds in the present study.
It is of interest to compare the kinetics and equilibria of cis/
trans isomerization of the secondary amide peptide bonds to
those of the much more abundant and well-studied Xaa-Pro
tertiary amide peptide bonds. The population of the cis
conformation of Xaa-Pro peptide bonds is typically in the
5-20% range for linear proline-containing peptides, while the
population of the cis conformation in cyclic, disulde-containing
peptides covers a much wider range, depending on the amino
acid sequence and size of the disulde-containing ring, e.g.,
from 1.4% for the Cys-Pro peptide bond of the disulde form
of Ac-Cys-Pro-Thr-Cys-NH
2
to 77% for the Cys-Pro peptide
bond of the disulde form of Ac-Cys-Pro-Phe-Ala-Ala-Ala-Cys-
NH
2
.
24
The much higher population of the cis isomer of Xaa-
Pro tertiary amide peptide bonds, as compared to the Phe-Ala,
Tyr-Ala, Ala-Phe, and Ala-Tyr secondary amide peptide bonds
in linear peptides, is due to a combination of increased kinetic
stability of the cis conformation and decreased kinetic stability
of the trans conformation of the Xaa-Pro peptide bond. For
example, the average rate constant for cis-to-trans interchange
for the Xaa-Pro peptide bond in 12 linear peptides, where Xaa
) Cys or Gly, is 0.047 s
-1
, as compared to the rate constants
for trans-to-cis interchange in Table 2 that are some 8-43 times
faster, while the average rate constant for trans-to-cis interchange
for the 12 Xaa-Pro peptide bonds is 6.6 10
-3
s
-1
, as compared
to the values for k
tc
in Table 2 which range from 4 to 19 times
slower.
24,25
The increased kinetic stability of the trans isomer
of the secondary amide peptide bonds, relative to that of the
Xaa-Pro peptide bonds, is due to less steric interaction between
the proton on the amide nitrogen of the secondary amide peptide
bonds and the side chain of the adjacent amino acid as compared
to steric interactions between the side chain of Xaa and the
-CH
2
group of the proline ring in the trans conformation of
the Xaa-Pro peptide bond. Likewise, the increased kinetic
stability of the cis conformation of the Xaa-Pro peptide bond
is due to less steric interaction between the side chain of Xaa
and the constrained proline ring, as compared to steric interac-
tions between the two side chains of the Phe-Ala, Ala-Phe, Tyr-
Ala, and Ala-Tyr secondary amide peptide bonds.
Conclusions
The results presented here signicantly increase the quantita-
tive data available on the kinetics and equilibria of cis-trans
isomerization of secondary amide bonds in peptides. The results
indicate the degree of conformational heterogeneity along the
backbones of peptides, and presumably unfolded proteins, and
the time scales of interchange among the conformational
isomers. A signicant nding is that, although cyclization by
disulde bond formation imposes conformational constraints on
the peptide backbones, cyclization has relatively little affect on
the population of the sterically less-favored cis conformation
and the rates of cis-trans isomerization of the Xaa-Yaa
secondary amide peptide bonds. This suggests that the kinetics
and equilibria of cis-trans isomerization of the Xaa-Yaa peptide
bonds are governed primarily by factors localized to the
dipeptide sequence.
Figure 4. Integrated intensity of the resonance for the Ala-methyl
protons of the cis isomer of peptide 2b as a function of the exchange
time t in the inversion-magnetization transfer pulse sequence. The
sample was 22 mM peptide 2b in 90% H
2
O/10% D
2
O at pH 2.91 and
25 C. The smooth curve through the points is the theoretical curve
obtained by nonlinear least-squares analysis of the data.
Isomerization of Secondary Amide Peptide Bonds J. Phys. Chem. B, Vol. 114, No. 9, 2010 3391
Acknowledgment. This work was supported in part by the
University of California, Riverside. M.I. was supported by the
Medical Scholars Summer Research Program, University of
California, Riverside. The authors thank Dan Borchardt for
assistance with the NMR measurements.
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