peptide bond isomerization
TRANSCRIPT
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NMR characterization of peptide bonds in linear and cyclic peptides
Darrien James and Markus Germann
Georgia State University, Atlanta, GA 30303
Abstract
NMR spectroscopy is used to study the peptide bond conformations in the linear and
cyclic forms of the tetrapeptide IGGN. Five minor peptide bond conformers were
discovered in the linear peptide. However, only a G3 peptide bond minor conformer was
observed for the cyclic tetrapeptide. At 293K, 0.41 % cis G-3 peptide bond isomer
population was detected for the linear tetrapeptide. The trans to cis isomerization of the
G-3 peptide bond is exothermic. The enthalpy of the process is 35.5 kJ/mol. It was also
found that the rate of the isomerization increases with increasing temperature.
Introduction
Peptide bonds form the backbone of protein chemistry. The amino acid sequence of a
protein contains important folding information. These unique folding patterns define the
function of the protein.1 A peptide bond has partial double bond character rendering the
bond planar. The electrons of the carbonyl bond are delocalized via resonance (figure
1),consequently, the bond is rigid but some rotation is possible.2 This characteristic
allows us to study the cis (Z) /trans (E) isomerization at peptide bonds.3NMR
spectroscopy can be employed in the study of the conformational change of peptide
bonds. 4
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Figure 1. Illustration of the peptide bond resonance.
Currently, most academic and pharmaceutical labs study peptides in their trans form. Our
research in cis peptide bond analysis could provide the foundation for a more holistic
study of peptides. Moreover, most small peptide drugs have trans peptide bonds.
Therefore, investigating the cis isomer will further diversify peptide drug chemistry.
Proteases naturally metabolize trans peptide bonds. Therefore, peptide drugs with their
peptide bonds locked in the cis conformation could be less susceptible to enzymatic
degradation. This innovation could potentially lead to a substantial improvement in the
bioavailability of peptide drugs.
Prolyl peptide bonds have been studied extensively. Proline residues are common at the
edge of beta strands and as the 1st residue in alpha helices. In a typical peptide bond the
trans isomer is preferred greatly over the cis isomer. However, the trans peptide bond is
only slightly preferred in a prolyl peptide bonds. This is due to the fact that the nitrogen
of proline is bonded to two tetrahedral carbon atoms.5
The proline cis isomer population of an unstructured peptide is 5%-50%. 6 The amount is
dependent on the amino acid that is adjacent to the proline. Prolines flanked by aromatic
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residues have higher cis content when compared to prolines flanked by residues of other
amino acids. This is due the steric restrictions generated by the large aromatic side chains
of tyrosine, tryptophan, and phenylalanine. Reimer found that the unstructured
oligopeptide acetyl-Ala-Xaa-Pro-Ala-Lys-NH2 showed proline cis populations were
highest for Trp-Pro peptide bonds, 38% and lowest for Pro-Pro, 6%. 7
While, researchers studying non-prolyl peptide bonds reporta cis secondary amide
peptide bond population ranging from 0.1% -1.00% for the following peptides: Ala-Tyr,
Tyr-Ala, Phe-Ala, Ala-Phe, Ala-Ala-Tyr, Ala-Ala-Tyr-Ala and Ala-Ala-Tyr-Ala-Ala.The cis-trans isomerization was quantified by NMR line shape analysis and
magnetization- transfer experiments.
The 1H NMR analysis of Ala-Tyr was carried out at different temperatures. The chemical
shifts for the cis and trans methyl protons of alanine in Ala-Tyr were recorded at 369 and
295K. The first run was performed by gradually raising the temperature to 369K. The line
broadening and signal shifts increased gradually.At 295K, the line shapes and signal
ratios of the alanine methyl protons were identical to those observed at 369K. New
signals were observed as time progressed at higher temperatures. However, unlike the cis
and trans methyl protons of alanine, these new minor conformers, due to partial
decomposition products, showed no reversibility. Consequently, Scherer et al. were able
to infer that the conformational isomerism of the dipeptide Ala-Tyr rendered the alanine
methyl group chemically different in both conformers. Exchange spectroscopy provided
further evidence of two inter-converting Ala-Tyr species.Thereafter, Scherer uses this
evidence and data as a model to study the secondary peptide bond isomerization of the
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Ala-Xaa moiety in all the peptides mentioned above. It was found that the cis populations
were dependent on chain length and ionization state of the peptides. 8Similarly, our
research involves the study of peptide bond conformations. However, our efforts are
focused on the cis (Z) / trans (E) isomerization at the primary peptide and not the
secondary peptide bond.1H and 13C NMR spectroscopy are used to determine the
population of the cis isomer present in a solution of the linear tetrapeptide with sequence
IGGN. NMR is also used to explore if conditions can be found that increase the
population of the cis isomer. NMR along with molecular modeling software is used to
determine the structure of the cyclic peptide IGGN. It is believed that all the peptidebonds in the cyclic peptide are in the trans form. This is due to the conformational
restriction produced by the ring.
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STRUCTURES OF THE LINEAR AND CYCLIC TETRAPEPTIDES I1-G2-G3-N4
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Procedure
Instrumentation
All NMR experiments were performed on a Bruker Avance 500 MHz spectrometer operating at a
500.18 MHz proton frequency. A 5 mm TBI probe head was used throughout the study.
Assignment
The assignments for both peptides were determined with 1-dimensional and 2-
dimensional NMR experiments. These techniques include 1D proton NMR, correlation
spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear single
quantum coherence spectroscopy (HSQC), nuclear overhauser effect spectroscopy
(NOESY) and heteronuclear multiple bond correlation spectroscopy (HMBC).
COSY experiments provided connectivity information for neighboring protons separated
by 3 bonds. The TOCSY experiment provided further connectivity information going
down the amino acid chain. In all TOCSY experiments the mixing time was set to 80 ms.
The NOESY technique was used to assign resonances that could not be assigned by other
experiments. The experiment requires that the atoms are close in space. The atoms must
be within 5-6 .9 The technique was also used to support other chemical shift
assignments obtained using correlation experiments. The NOESY mixing times that were
used in the cyclic and linear chemical shift assignments were 50, 75, 150 and 300 ms. A
50 ms experiment was performed on the linear peptide while a 75 ms experiment was
performed on the cyclic peptide.
The following heteronuclear experiments were performed to obtain data on C-H
connectivity, long-range carbon to hydrogen coherence and nitrogen chemical shifts. The
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13C HSQC provided data on C-H connectivity. While, the 15N HSQC provided the
nitrogen chemical shifts for the cyclic peptide. The HSQC, was indirectly referenced
using the method outlined by Wishart et. al.10 The HMBC technique facilitates the
determination of long range (two and three bond) H- heteronucleus connectivity. It was
employed in the determination of long-range carbon to hydrogen coherence in the cyclic
peptide. The experiment was used to assign the carbonyl chemical shifts for the cyclic
peptide.11
Linear peptide
A 6.5 mM solution of the linear peptide was prepared by dissolving 2.6 mg of the peptide
in 1 mL of DMSO. Initially, 1D andNOE spectra were used to detect minor conformers
of all four peptide bonds. In the NOE spectra, each peptide bond had 1 or more exchange
peaks with smaller diagonal peaks within the amide region (figure 3). These peaks were
identified as the minor conformers of the peptide bonds.The signal intensities of the
peaks were measured and tabulated. However, the G-3 peptide bond was chosen for
further 1D peptide bond characterization studies because it had the strongest NOE
exchange peaks at a temperature of 298K and a mixing time of 300 ms. Four1H NMR
experiments were performed at the following temperatures 293, 303, 308, and 313K. The
peak areas for the major and minor conformers of the G-3 peptide bond were then
calculated using the integration function. The signal of the major conformer was used as
the calibration peak. It was assigned an area of 1 then the minor conformer peak was
integrated. These areas were used to determine the equilibrium constant for the
isomerization at each temperature. Thereafter, the free energy, enthalpy and entropy
changes involved in the isomerization were calculated from the equilibrium constants.
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Cyclic Peptide
A6.3mM solution of the cyclic peptide was prepared by dissolving 1.8 mg of the
peptide in 0.6 mL of DMSO. Thereafter, a more concentrated 30mM solution was made
to obtain better signal to noise in NMR experiments. The Sparky program was used to
assign and integrate NOE cross peaks.12 Cross peak intensities obtained from the distance
map were then used to calculate the distances between all hydrogens that produced NOE
signals. Several models of the cyclic tetrapeptide were constructed using PCModel. The
first had all peptide bonds in the cis conformation, while the other two models each had 1of the glycine peptide bonds in the cis conformation. There after a series of models were
constructed in which the carbonyl orientations were varied.
Results
The chemical shift assignments for the linear and cyclic peptides were mostly routine.
However, differentiating the 2 glycines was more difficult. NOE experiments allowed for
the differentiation between the glycines. It was also interesting that the alpha hydrogens
of the glycines were degenerate in the linear but not in the cyclic peptide. This provided
evidence of the cyclic peptides conformational restriction. Also, for resonances with 2
protons such as alpha hydrogens of the glycines, the hydrogen with the lower chemical
shift was named alpha-1. Similarly, for the beta and gamma hydrogens of isoleucine, the
hydrogens with the lower chemical shift were named beta-1 and gamma-1. The same
convention was used for naming the beta hydrogens of asparagine. The chemical shift
assignments are in the tables below.
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THE LINEAR TETRAPEPTIDE I1-G2-G3-N4
Table 1. NMR chemical shifts of the linear peptide hydrogens and carbons in DMSO at 298K.
THE CYCLIC TETRAPEPTIDE I1-G2-G3-N4
Ile (I1) Gly (G2) Gly (G3) Asp (N4)1H 13C 15N 1H 13C 15N 1H 13C 15N 1H 13C 15N
N-H 7.68 7.75 8.67 8.32
H1 3.92 58.1 3.51 42.0 3.58 43.0 4.46 50.0
H2 3.92 3.72
H1 1.81 36.0 2.44 36.0
H2 2.39
H1 1.14 25.0
H2 1.47
H' 0.81 15.0
H 0.78 10.0
NH2-1 6.93
NH2-2 7.33
C=O 171.7 170.9 169.0 171.1
Amide N 110.0 99.6 104.2 113.7
N
Table 2. NMR chemical shifts of the cyclic peptide hydrogens and carbons in DMSO at 298K.
Ile (I1) Gly (G2) Gly (G3) Asp (N4)1H 13C 1H 13C 1H 13C 1H 13C
N-H 7.92 8.25 8.08 8.03
H1 4.12 57.0 3.71 42.0 3.68 42.0 4.46 50.0
H2 3.71 3.68
H1 1.40 24.3 2.50 37.0
H2 2.39
H1 1.08 24.3
H2 1.08
H' 0.81 15.0
H 0.80 11.0
NH2-1 6.85
NH2-2 7.27
Term. NH2-1 7.04
Term. NH2-2 7.10
Acetyl 1.87 22.2
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Figure 2.1H NMR spectra of the linear tetrapeptide within the amide proton region at298K in DMSO. The intensity of the G-3 minor peak was far lower than that of the major N-H signals as aresult the two spectra with different peak height scales were superimposed.
Figure 3. NOESY spectra of the amide protons of the linear tetrapeptide at 308 K witha mixing time of 150 ms in DMSO.
G - 3 minor
I 1
G3G2
N4
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Minor conformers were detected for all four peptide bonds. Interestingly, two exchange
peaks were observed for the G3 peptide bond. However, only 1 was identified as being
due to a minor conformer. This G3 minor conformer is labeled in the spectrum above.
Figure 4. NOESY spectra of the amide protons of the cyclic tetrapeptide at 308 K with
a mixing time of 150 ms in DMSO.
In the exchange spectrum above the minor conformer of the G3 N-H proton of the cyclic
tetrapeptide is shown. Though the diagonal peak of the minor conformer was not
completely resolved, the cross peaks in both dimensions provide evidence that there is a
minor conformer of the G3 N-H proton with a chemical shift of 7.95 ppm. It is
hypothesized that this minor conformer is the cis G-3 peptide bond. Since, the
tetrapeptide has very little conformational mobility.
G-3
N-4
I-1
G-2
exchange peak
exchange peak
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STRUCTURE OF N-METHYLACETAMIDE
TRANS CIS
EMM = -7.43 kcal/mol EMM = -0.035 kcal/mol
QM Chemical Shift Calculations (ppm)
Trans Cis
4.89 4.66
Table 3. Spartan QM Chemical shift calculations for the cis and trans conformations of a peptide bondamide hydrogen in n-methylacetamide using the B3LYP6-31G** density function.
The quantum mechanical calculations above supported the 1D chemical shift order of the
G-3 N-H major and minor conformers (figure 2). Both procedures showed that the
chemical shift of the major conformer was higher. The minimization energies were
calculated in Hyperchem. The MM+ force field and the steepest decent minimization
algorithm were employed.
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Figure 5. Graph showing the temperature dependence of the cis G-3 peptide bond abundance. The abovecurve illustrates that the amount of the cis isomer decreases with increasing temperature. The R2 value forthe curve is 0.9391.
T (K) % Cis Fraction Cis Fraction Trans K ln (K) 1/T G a H S b
(1/K) (J/mol) (J/mol) J/mol-K
293 0.41 0.0041 0.99996 4.1 x10-3
-5.50 3.41 x10-3
13399 -35528 -205.2298 0.39 0.0039 0.99996 3.9 x10-3 -5.55 3.36 x10-3 13751 -35528 -203.6
303 0.27 0.0027 0.99997 2.7 x10-3 -5.91 3.30 x10-3 14889 -35528 -204.7
308 0.21 0.0021 0.99998 2.1 x10-3 -6.17 3.25 x10-3 15801 -35528 -204.9
a From the free energy equation G = -RTlnKb From the free energy equation G = H-TS
Table 4. Thermodynamic data for the G-3 peptide bond isomerization in DMSO.
The calculated Gibbs free energies were all positive therefore the isomerization is
nonspontaneous between 293 and 308K. It was found that the percentage cis G-3 NH
increased as the temperature was lowered. Therefore, the G3 trans cis isomerization
for the linear tetrapeptide IGGN is an exothermic process. The calculated enthalpy for the
G3 trans cis isomerization was 35.5 kJ/mol.
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Figure 6. G-3 peptide bond Isomerization: Van Hoff Plot: ln (K) as a function of reciprocal temperature.
The following equation, lnK = -H/RT + S/R, was used to construct a Van Hoff plot
for the isomerization. The lnK was the y term while 1/T was the x term. The slope =
H/R= 4273.6 K. This value was then was then multiplied by the gas constant to give
the enthalpy of the isomerization, H= -35.5 kJ/mol. Van Hoff Curve Equation: y =
4286.4x - 20.047 R2 = 0.9297.
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Table 5. Intensity of NOESY spectra exchange peaks for the major and minor conformations of the peptideamide bond protons. All of the above NOESY experiments were done in DMSO.
Temperature(K)
Mixing Time(ms)
AmideProton
MajorPeak
ExchangePeak A
ExchangePeak B
G-2 8.8x106
7500
293 150 G-3 9.2x10
6
8400 7700
N-4 7.8x106
6400
G-2 10.0x106
9592
298 50 G-3 11.0x106
8800 7012
N-4 9.0x106
6212
G-2 8.6x106
12000
298 150 G-3 8.6x106
12000 10000
N-4 7.6x106
11400
G-2 12.5x106
32000
298 300 G-3 12.6x106
33000 26000
N-4 12.0x106
24000
G-2 7.5x106
20540
308 150 G-3 8.2x106
21000 21000
N-4 7.2x106
18000
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This NOESY analysis was done to get the rate of the trans-cis isomerization.The peak
intensities were measured using both the NOESY and 1D experiments. The exchange
peaks labeled A are for the trans-cis isomerization of I1, G2, G3 and N4 amide hydrogens
with the corresponding diagonal peaks. The exchange peak labeled B was only detected
for G-3 peptidebond. Currently, we are not sure of the identity of the diagonal peak that
corresponds to the exchange peak labeled B. It could be 1 minor form inducing another
minor form.
Isomerization Rates
mt (s) Xa Xb Iaa Ibb Iab Iba r k ct (s-1) k tc (s-1)
0.01 0.998 0.0021 6.70x107 1.41 x105 33000 14000 10.9 120.1 0.252
0.02 0.998 0.0021 6.00 x107 1.26 x105 39000 38000 5.54 72.02 0.151
0.03 0.998 0.0021 6.10 x107 1.28 x105 73000 41000 3.49 60.06 0.126
0.05 0.998 0.0021 6.00 x107 1.26 x105 70000 70000 2.60 44.98 0.095
Table 6. G3 peptide bond isomerization data.
A model developed by Perrinet .al was employed to calculate the rates of isomerization
of the G3 peptide bond at 308K. The formula below shows how the data in the table
above was computed.13
Eqn. 1
Eqn. 2
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Perrins method proved to be ineffective for this system because the relative population
difference was too vast. However, other appropriate models will be tested and
implemented.
Rates analysis
Seven NOE experiments with different mixing times were carried out at 308 K to
examine the intensity change of the cross peaks between the major N-H diagonal peaks
and the smaller cis conformer peaks (figure 3labeled NOE) The mixing times were 50
ms, 100 ms, 250 ms, 500 ms, 750 ms, 1000 ms and 1500 ms.
The diagonal peak intensities all decreased as the mixing time was increased. While, the
cross peak intensities increased to a maximum at a specific mixing time, thereafter, their
intensities decreased. The following curves summarize the diagonal and cross peak
development as a function of mixing time.
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Figure 7. NOE Intensity of the I1 N-H diagonal peak as a function of mixing time.
Figure 8. NOE Intensity of the G2 N-H diagonal peak as a function of mixing time.
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Figure 9. NOE Intensity of the G-3 N-H diagonal peak as a function of mixing time.
Figure 10. NOE Intensity of the N-4 N-H diagonal peak as a function of mixing time.
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Figure 11. NOE intensity of I1 N-H exchange peak A as a function of mixing time.
Figure 12. NOE intensity of I1 N-H exchange peak B as a function of mixing time.
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Figure 13. NOE intensity of G2 N-H exchange peak A as a function of mixing time.
Figure 14. NOE intensity of G2 N-H exchange peak B as a function of mixing time.
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Figure 15. NOE intensity of G-3 N-H exchange peak 1A as a function of mixing time.
Figure 16. NOE intensity of G-3 N-H exchange peak 1B as a function of mixing time.
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Figure 17. NOE intensity of G-3 N-H exchange peak 2A as a function of mixing time.
Figure 18. NOE intensity of G-3 N-H exchange peak 2B as a function of mixing time.
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Figure 19. NOE Intensity of the N4 N-H exchange peak A as a function of mixing time.
Figure 20. NOE Intensity of the N4 N-H exchange peak B as a function of mixing time.
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Figure 21. T1 Inversion Recovery plot of the G-3 N-H minor form diagonal peak as a function of delay ().
The 1D 1H NMR intensity of the minor form of the G-3 NH resonance obtained from a
T1 IR experiment was plotted as a function of delay to determine the spin-spin lattice
relaxation time (T1). The T1 value was obtained from a curve fittingto the general
equationy= m1*(1-2*e-x/m2). The T1 value for the minor form of G3 N-H was 583 ms.
This value is close to the average T1 obtained for the G3 N-H major conformer, 623 ms.
The table below summarizes the T1 values obtained for the N-H protons at 293, 298, 303,and 308K.
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Temperature
(K) N-H T1(ms)
I1 600G2 538
293 G3 627
N4 600
I1 597
G2 528
298 G3 615
N4 582
I1 607G2 534
303 G3 618
N4 584
I1 624
G2 537
308 G3 632
N4 597
Table 7. Spin-spin lattice relaxation times for the N-H protons of the linear tetrapeptide at varioustemperatures.
Structural Elucidation of the Cyclic tetrapeptide IGGN
NOESY spectra were used to determine the distance between the hydrogen atoms in the
cyclic tetrapeptide I1-G2-G3-N4. The distance between NH2-1- NH2-2 amide hydrogens
of the asparagine was used as the reference distance. The table below shows the NH2-1-
NH2-2 distance measured from a model of the cyclic peptide constructed in PC Model,
alongwith, the NOE cross peak intensities forNH2-1- NH2-2 at three mixing times.
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The NH2 -1- NH2-2 amide reference distance, the NOE cross peaks intensities forNH2
-1- NH2-2 and the individual resonances were substituted into the equation below to
calculate all the tabulated distances. All reported distances are in angstroms ().
Ia / Ib = rb6 / ra
6 Eqn. 3
Mixing Time (ms) NOE Intensity Distance ()75 5.64 x 10
6 1.65
150 7.47 x 106 1.65
300 6.38 x 106 1.65
Table 8. NOE reference data collected at three different mixing times.
NH-NH
N-H
N-H
Table 9. NH-NH distances calculated at 3 different mixing times. The abbreviation ne means there was no
peak.
I1 G2 G3 N4
75 1.9 ne 2.6
150 I1 2.0 5.2 2.6
300 2.1 4.5 2.5
75 3.2 3.9
150 G2 3.1 3.7
300 3.0 3.7
75 3.1
150 G3 3.1
300 3.2
75
150 N4300
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H- NH
N-H H
Table 10. H-NH distances calculated at 3 different mixing times. The following abbreviations are used
in the above tabulation: ne- none: there were no cross peaks, n-low S/N, c-coupling: spin coupling peak
distortion, and vc- very close to the diagonal: the NOE cross peak is too close to the diagonal to obtain
accurate volumes. *+I1H and G2H2 are isochronous; both hydrogens have the same chemical shift, as a
result, molecular modeling applications were employed to differentiate the alpha hydrogens. The volumes
obtained for these resonances with identical chemical shifts were compared with model distances to
decipher the cross peaks.
H s I1 G2 G3 N4
75 2.7 +ne ne ne
150 I1 2.5 +ne ne ne
300 3.92 2.7 +ne ne 3.9
75 1-n 2-n 1- n 2-2.4+ 1-n, vc *2-2.3 1-ne 2-ne
150 G2 1-n 2-n 1-2.4 2-2.3+ 1-n *2-2.3 1-ne 2-ne
300 3.51, 3.92 1-n 2-n 1-2.3 2-2.2+ 1-n *2-2.2 1-ne 2-ne
75 1-ne 2- n 1-n 2-n 1- n, vc 2- 2.6 1-3.0 2-2.3
150 G3 1-ne 2-3.5 1-n 2-n 1-2.2 2- 2.5 1-2.8 2-2.3
300 3.59,3.72 1-ne 2-3.5 1-n 2-n 1-2.2 2- 2.7 1-3.1 2-2.2
75 2.7 3.8 ne c
150 N4 2.6 4.4 ne 2.4
300 4.46 2.6 4.2 ne 2.4
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H- All other Resonances
All other Resonances
H
Table 11. H -All other resonance distances calculated at 3 different mixing times. * I1H and G2H2 are
isochronous. However, model evidence supports these distances are G2H1- G2H2. G31, G32 and
G21 all have cross peaks with I1' however these very weak peaks were in the noise.
H All other Resonances
All other Resonances H
I1 G2 G3 N4
75 H- 2.8 1-ps 2- 2.5 '- 2.5 ne ne
150 I1 H- 2.8 1-2.9 2- 2.6 '-2.4 ne ne
300 H- 2.8 1- 2.9 2- 2.4 '-2.4 ne ne
75 ne ne H- 2.7 NH2-1- 4.2 NH2-2- 2.9
150 N41 ne ne H- 2.8 NH2-1- 4.3 NH2-2- 2.9
300 ne ne H- 2.5 NH2-1- 3.6 NH2-2- 2.8
75 ne ne H- 2.4 NH2-1- n NH2-2- n
150 N42 ne ne H- 2.2 NH2-1- 4.2 NH2-2- 4.2
300 ne ne H- 2.6 NH2-1- 4.2 NH2-2- 4.2
Table 12. All H distances calculated at 3 different mixing times. The I1 and I1' resonances had similar
chemical shifts; they are 0.78 and 0.81 respectively. The N41 - N42 cross peaks were very close to the
diagonal and the peaks volumes were inconsistent.
I1 G2 G3 N4
75 -2.8 '-2.7 1- 3.2 2-3.7 ne ne ne
150 I1 -2.8 '-2.5 1- 3.1 2-3.7 ne ne ne
300 - 2.8 '-2.5 1- 3.0 2-3.6 ne ne ne
75 ne H- 1.8 ne ne
150 G2 ne H- 1.8 ne ne
300 ne H- 1.8 ne ne
75 ne ne H- 1.6 ne
150 G3 ne ne H- 1.7 ne
300 ne ne H- 1.7 ne
75 ne ne ne ne
150 N4 ne ne ne ne
300 ne ne ne ne
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NH All other Resonances
All other Resonances N-H
Table 13. N-H- all other resonances distances calculated at 3 different mixing times.
The tables below summarize the N-H to N-H distance data obtained by varying the
backbone carbonyl orientation of each amino acid in various models of the cyclic peptide.
The models that were closest to the experimental distances obtained from the NOE
experiment were G2 carbonyl down others up (table 18) and the G3 carbonyl down others
up (table 17). This data suggests that the actual structure of the peptide could be similar
to 1 of these models.
N-H to N-H Model () NOE (mt=150ms) () Absolute Difference
I1-G2 2.2 2.0 0.2
G2-G3 2.5 3.1 0.6
G3-N4 2.6 3.1 0.5
I1-G3 3.6 5.2 1.6
I1-N4 2.8 2.6 0.2
G2-N4 3.6 3.7 0.1
Table 14. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with allcarbonyls pointing downward.
N-H I1 G2 G3 N475 -2.5 1- 3.1 2-3.3 y'-3.4 1- 3.8 2- 3.8
150 I1 N-H - 2.5 1-3.1 y2-3.2 y'-3.3 1- 3.6 2- 3.6
300 -2.4 1- 2.9 y2-3.1 y'-3.1 1- 3.4 2- 3.3
75 - 3.0 '- 3.3
150 G2 N-H - 2.9 '- 3.3
300 - 2.7 '- 4.1
75
150 G3 N-H300
75 1- 2.6 2- 3.1
150 N4 N-H 1- 2.7 2- 3.1
300 1- 2.6 2- 3.0
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N-H to N-H Model () NOE (mt=150ms) () Absolute Difference
I1-G2 4.1 2.0 2.1
G2-G3 4.1 3.1 1.0
G3-N4 4.1 3.1 1.0
I1-G3 4.3 5.2 0.9
I1-N4 4.1 2.6 1.5
G2-N4 4.3 3.7 0.6
Table 15. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with alternatingcarbonyls.
N-H to N-H Model () NOE (mt =150ms) () Absolute DifferenceI1-G2 3.5 2.0 1.5
G2-G3 2.7 3.1 0.4
G3-N4 2.6 3.1 0.5
I1-G3 4.6 5.2 0.6
I1-N4 4.0 2.6 1.4
G2-N4 3.8 3.7 0.1
Table 16. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN withN4 carbonylpointing down and all other carbonyls pointing up.
N-H to N-H Model () NOE (mt=150ms) () Absolute Difference
I1-G2 2.4 2.0 0.4
G2-G3 2.4 3.1 0.7
G3-N4 4.1 3.1 1.0
I1-G3 4.4 5.2 0.8
I1-N4 4.0 2.6 1.4
G2-N4 3.7 3.7 0.0
Table 17. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN withG3 carbonyl
down and all other carbonyls pointing up.
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N-H to N-H Model () NOE (mt=150ms) () Absolute Difference
I1-G2 2.1 2.0 0.1
G2-G3 4.0 3.1 0.9
G3-N4 3.9 3.1 0.8
I1-G3 4.3 5.2 0.9
I1-N4 2.8 2.6 0.2
G2-N4 4.0 3.7 0.3
Table 18. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with G2 carbonyldown and all other carbonyls pointing up.
N-H to N-H Model () NOE (mt=150ms) () Absolute Difference
I1-G2 4.2 2.0 2.2
G2-G3 3.8 3.1 0.7
G3-N4 3.0 3.1 0.1I1-G3 3.7 5.2 1.5
I1-N4 2.8 2.6 0.2
G2-N4 5.5 3.7 1.8
Table 19. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN withI1 carbonyldown and all other carbonyls pointing up.
N-H to N-H Model () NOE (mt=150ms) () Absolute Difference
I1-G2 3.6 2.0 1.6G2-G3 3.2 3.1 0.1
G3-N4 3.9 3.1 0.8
I1-G3 5.5 5.2 0.3
I1-N4 3.0 2.6 0.4
G2-N4 4.1 3.7 0.4
Table 20. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with G3 and N4carbonyls down and all other carbonyls pointing up .
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N-H to N-H Model () NOE (mt=150ms) () Absolute Difference
I1-G2 2.4 2.0 0.4
G2-G3 3.1 3.1 0.0
G3-N4 2.9 3.1 0.2
I1-G3 4.6 5.2 0.6
I1-N4 4.2 2.6 1.6
G2-N4 4.2 3.7 0.5
Table 21. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with N4 and I1carbonyls down and all other carbonyls pointing up .
Figure 22. A model of the cyclic tetrapeptide IGGN with G2 carbonyl down and other carbonyls up. Thismodel has the best agreement with experimental N-H to N-H distances.
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Discussion
In the linear peptide, the concentration of the cis peptide bond isomers increased with
increasing temperature. This result is consistent with earlier work conducted, which
showed that the concentration of secondary cis peptide bonds increased with increasing
temperatures for small peptides with the Ala-Xaa moiety.
Remarkably, thermodynamic calculations revealed that the enthalpy of the G-3 peptide
bond trans to cis isomerization is exothermic. It was hypothesized that the G3 peptide
bond trans-cis isomerization would be an endothermic process since the cis conformer
produces steric clashes between the oxygen and the hydrogen of the amide group.Furthermore, from the free energy equation, G = H-TS, the negative enthalpy and the
positive free energy shows that the reaction is entropy driven.
While, the NOE rates data plots produced the expected trend; an exponential decay in N-
H diagonal peak intensity as a function of mixing time and an increase in the
cross peak intensities until a maximum intensity was reached, thereafter, the cross peak
intensities decreased. The T1 inversion recovery showed that the spin-spin lattice
relaxation of all four N-H protons were similar. They ranged from 527- 632 ms.
The distance data obtained from the NOE experiments were reliable and made spatial
sense. There was also very good agreement in distances among the 3 mixing times.
Therefore, spin diffusion effects were minimized. The group of cyclic models with varied
backbone carbonyl orientation provided further insight on the structure of the cyclic
tetrapeptide IGGN. Our research has shown that it is possible to directly study peptide
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bond isomerization complementing earlier work which placed more emphasis on the
secondary amide peptide bond orientation.
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References
1. Branden, C.Introduction to Protein Structure. Garland. NY. 1998, 89-91.
2. Pauling, L. The Nature of the Chemical Bond. Cornell. NY. 1948, 10-14.
3. Berg, J.Biochemistry. W. H. Freeman. NY. 2006, 37-38.
4. Balci, M.Basic 1H- and13C- NMR Spectroscopy. Esevier. Boston. 2005, 3-4.
5. Eberhardt, E., Loh, S., Raines, R. Tetrahedron Letters. 1993, 34, 3055-3056.
6. Li, P.S., Chen, E., Shulin, S., Asher, A. J. Am. Chem. Soc. 1997, 119, 1116.
7. Reimer, U., Elmokdad, N., Schutkowski, M., Fischer, G.,Biochemistry, 1997, 36,13802.
8. Scherer, G.J Amer. Chem. Soc. 1998, 120, 5568-5574.
9. Wtrich, Kurt.NMR of Proteins and Nucleic Acids. John Wiley. 1986, 117-120.
10. Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E.,Markley, J. L., Sykes, B. D.,J. Biomol. NMR. 1995, 6, 135-140.
11. Homans, S.A Dictionary of NMR. Clarendon Press. Oxford. 1992, 148-150
12. Goddard, T. D. Sparky 3. University of California, San Francisco.
13. Perrin, C., Gipe, R.J. Am. Chem. Soc. 1984,106, 4036.
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