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www.sciencemag.org/cgi/content/full/332/6034/1206/DC1
Supporting Online Material for
Residue-Specific Vibrational Echoes Yield 3D Structures of a Transmembrane Helix Dimer
Amanda Remorino, Ivan V. Korendovych, Yibing Wu, William F. DeGrado, Robin M. Hochstrasser*
*To whom correspondence should be addressed E-mail: [email protected]
Published 3 June 2011, Science 332, 1206 (2011)
DOI: 10.1126/science.1202997
This PDF file includes:
Materials and Methods SOM Text Figs. S1 to S10 Tables S1 and S2 Full Reference List
1
Supporting Online Material
Content Summary:
1. Material and methods
2. FTIR data
3. 2D-IR data
4. Model to extract the coupling constants
5. Fit of the FTIR and 2D-IR spectra
6. Structure determination
1. Materials and Methods:
Peptide synthesis. Twelve different peptides were synthesized by introducing 13
C=18
O labels on
each of the eleven residues L10 to L20 of CGGKPIWWVL10VGVLGGLLLL20TILVLAMWKK
in addition to an unlabeled peptide. N-Fmoc-1-13
C=18
O labeled amino acids (Gly, Leu, Val) were
prepared from the corresponding N-Fmoc-1-13
C labeled amino acids (Cambridge Isotope
Laboratories, Andover, MA) and H218
O according to the literature procedures (15) allowing for
at least 90% isotopic enrichment in 18
O, as evidenced by ESI-MS spectra of the products. The
TM portion of human IIb integrin (965-990) was additionally flanked with Lys residues and a
CGG linker. The peptides were synthesized on a PTI Symphony automated peptide synthesizer
using standard Fmoc protocols on a 0.05 mmol scale using a Fmoc-PAL-PEG -PS resin (Applied
Biosystems) with a substitution level of 0.21 mmol/g. Activation of the free amino acids (5 fold
excess) was achieved with 0.95 equiv (relative to the amino acid) excess of HATU in the
presence of 10 equiv of diisopropylethylamine (DIEA). The reaction solvent contains 25%
dimethylsulfoxide (DMSO) and 75% N-methylpyrrolidone (NMP) (HPLC grade, Aldrich). Side
chain deprotection and simultaneous cleavage from the resin was performed using a mixture of
trifluoroacetic acid (TFA)/triethylsilane/water/ethanedithiol (94:2.5:2.5:1 v/v) at room
temperature, for 3 hours. After filtration most of the solvent was evaporated using a stream of
2
N2. The crude peptides collected from precipitation with cold diethyl ether (Aldrich) were dried
in vacuo. The peptides were then purified on a preparative reverse phase HPLC system (Varian
ProStar 210) with a C4 preparative column (Vydac) using a linear gradient of buffer A (0.1%
TFA in Millipore water) and buffer B (6:3:1 2- propanol:acetonitrile:water) containing 0.1%
TFA. The identities of the purified peptides were confirmed by MALDI-TOF mass spectroscopy
on a Voyager Biospectrometry Workstation (PerSeptive Biosystems), and their purity was
assessed using HP1100 analytical HPLC system (Hewlett Packard) with an analytical C-4
column (Vydac) and a linear A/B gradient.
Stock solutions in trifluoroethanol (TFE) were prepared from the lyophilized powder. The
samples for 2D-IR measurements were prepared by mixing the stock of peptides with the 100
mM stock of dodecyl phosphatidylcholine (DPC), the solvent was removed in the stream of
nitrogen and the resulting films were dried in vacuo overnight to remove all of the organic
solvent leftover. The peptide-detergent film was then dissolved in D2O (20 mM phosphate
buffer, pH (uncorrected) 7.4) to the final concentration of peptide of 4 mM and the detergent of
200 mM.
Analytical Ultracentrifugation. Equilibrium sedimentation was used primarily to determine the
association state of the peptides and to provide an estimate of the association constants. The
experiments were performed in a Beckman XL-I analytical ultracentrifuge (Beckman Coulter)
using six-channel carbon-epoxy composite centerpieces at 25 °C. Peptides were co-dissolved in
TFE (Sigma) and DPC (Avanti Polar Lipids). The organic solvent was removed under reduced
pressure to generate a thin film of peptide/detergent mixture, which was then dissolved in buffer
previously determined to match the density of the detergent component (10 mM phosphate
buffer (pH= 7.4), containing 500 mM tris(2-carboxyethyl)phosphine (TCEP) in 52% D2O). The
final concentration of DPC is 15 mM in all of the samples. Samples were prepared in a total
peptide concentration of 38 µM. Data at different measurement speeds (35, 40, 45, 48 and 50
krpm) were analyzed by global curve-fitting of radial concentration gradients (measured using
optical absorption) to the sedimentation equilibrium equation for monomer-dimer equilibria
among the peptides included in the solution. Peptide partial specific volumes were calculated
using previously described methods (30) and residue molecular weights corrected for the 52%
D2O exchange expected for the density-matched buffer. The solvent density (1.059 g/ml) was
3
measured using a Paar densitometer. Sedimentation equilibrium data were fit using Igor Pro
(Wavemetrics) to the following equation:
Abs E ac0alexp 2
2RTMa (r
2 r02)
2a
c0a2
Kalexp
2
2RT2Ma (r
2 r02)
where E = baseline (zero concentration) absorbance, coa is the molar concentration of monomeric
IIb TM peptide at ro, ea is the molar extinction coefficient for IIb TM peptide at 280 nm, l is
the optical path length, =2*rpm, R= 8.3144 107 erg K
-1mole
-1 , T is temperature in K, Ma is
the buoyant molecular weight of monomeric IIb; Ka is the homodimeric dissociation constant for
IIb TM peptide.
Molecular weight was obtained from the buoyant molecular weight using:
Mw M(1 v_
)
where M is the buoyant molecular weight,
v_
is the partial specific volume and
is the solution
density.
Figure S1: Sedimentation equilibrium
profile at 280 nm of IIb TM peptide (38
µM) in density matched DPC micelles (15
mM) in phosphate buffer (10 mM, pH
7.4). The partial specific volume and the
solution density were fixed at 0.80057 mL/g
and 1.059 g/mL. The data was analyzed
using a global fitting routine. The molecular
weight was held at 3300 and the data were fit
to dissociation constant of 0.013
(peptide/detergent molar units).
4
FTIR Spectra. The FTIR spectra (Nicolet 6700) were corrected with a DPC and D2O
background subtraction. The second step involved the isolation of the isotopically substituted
amide I transitions from those of the 12
C=16
O main band. This was a difficult task given that in
the majority of the diluted cases, no peaks could be identified in the 13
C=18
O region. For this
purpose, the isotopically substituted region (1575-1615 cm-1
) was devoid of the 13
C=18
O amide I
transition known from the 2D-IR spectra and the remaining baseline was fitted with a Gaussian.
This Gaussian baseline was subtracted from the original spectrum resulting in the data shown in
Fig. 2 and the ones shown here. The accuracy of the subtractions was evaluated by comparison
of the obtained FTIR baseline to that of the background of the 2D-IR spectra.
2D-IR Spectra. The 2D-IR spectra were obtained by methods previously detailed (6). The echo
signal field generated at frequency t following a sequence of three infrared pulses was
measured as a function of the initial coherence frequency, . Each 2D-IR spectrum of vs. t
is recorded at a particular choice of the waiting time delay, T, between the second and third
pulses. The positive maxima in the 2D-IR are displaced to higher t frequencies because of the
interference with the negative v=1→2 portion.
5
2. FTIR data:
The DPC and D2O subtracted FTIR spectra of all the 100% and 10% 13
C=18
O labeled samples
can be seen in Fig. S2.
Figure S2: Subtracted FTIR
spectra. FTIR spectra of (A)
100% and (B) 10% 13
C=18
O
labeled samples. The
differences in OD between
samples shown in panel A is
given mostly by the variability
in the amount of dissolution of
the peptides after
lyophilization. The amide I
main band peak appears at 1656
cm-1
and a secondary peak at ca. 1635 cm-1
is attributed to small amounts of exposed amide I modes (31). The
transition at approx 1675 cm-1
is trifluoro acetic acid (TFA) whose further removal was avoided to minimize
aggregation.
The normalized FTIR spectra are also presented in Fig. S3 in order to compare the line shapes of
all the different samples. For most of the 10% labeled samples no 13
C=18
O bands could be
observed in the FTIR spectra but were present in the 2D-IR spectra.
Figure S3: Normalized FTIR
spectra: Normalized FTIR
spectra of (A) 100% and (B) 10% 13
C=18
O labeled samples.
The procedure used to isolate these transitions is shown in Fig. S4.
Figure S4: FTIR spectra
background subtraction. Method
used to separate the 13
C=18
O band
from the tail of the main band for (A)
G12 100% and (B) 10% 13
C=18
O
labeled samples. The spectrum devoid
of the 13
C=18
O transition (full circles)
was fitted with a Gaussian (red) that
was subtracted from the full spectrum
(empty circles).
6
3. 2D-IR data:
An example of a 2D-IR spectrum showing the complete amide I region is presented in Fig. S5. In
particular the one for 100% 13
C=18
O labeled G12 is shown.
Figure S5: 2D-IR correlation spectrum of 100% 13
C=18
O labeled G12. The amide I main band
transition appears at =1656 cm-1
. At ca. =1635
cm-1
the amide I transition of hydrated residues can
be seen. The transition at ca. =1613 cm-1
is from 13
C=16
O that exists due to natural abundance of 13
C
and to incomplete conversion of G12 from 13
C=16
O
to 13
C=18
O (ca. 5%). All the amide I transitions
present a non diagonal peak at lower energies (ca.
10 cm-1
) that is constant with waiting time (T)
indicating that it is not produced by a chemical
exchange phenomenon.
The isotope labeled region of the 2DIR spectra of all the samples are presented in Fig. S6. The
population decay time (T1) of the main band (12
C=16
O residues) was calculated from transient
grating experiments. The intensity of the 13
C=18
O labeled peaks in the 2D-IR spectra decayed at
the same rate with waiting time (T).
7
8
Figure S6: 2D-IR correlation spectra. The isotope region of the 2D-IR correlation spectrum of (bottom) 10%,
(middle) 20% and (top) 100% 13
C=18
O labeled samples. The dashed yellow line indicates the peak frequency
assigned to 13
C=18
O used for the analysis.
4. Model to extract the coupling constants:
Neglecting the mixed mode anharmonicity the 5x5 hamiltonian is:
j
ji
i
j
i
H
22000
2200
02200
000
000
in the basis set of the local sites i,0 , 0,j , i2,0 , 0,2 j and ji, . The 0,i state represents
a one quantum excitation in a given labeled residue in one helix and the j,0 in the other
whereas 0,2i represents a two quanta excitation in one helix, j2,0 in the other and ji, a
one quantum excitation in each helix. The eigenvalues and eigenvectors were calculated
numerically for all the possible combinations of i and j. We can write the one quantum
eigenstates as:
0,2
sin,02
cos0,,0 22 jeiejcicii
ji
0,2
cos,02
sin0,,0 22 jeiejcicii
ji
Where ij 2tan and ie . We express the two quanta eigenstates in a symbolic
form as:
ijcjcicSS
ji
S
j
S
i ,0,22,0 22
ijcjcicSS
ji
S
j
S
i ,0,22,0 22
9
ijcjcicAA
ji
A
j
A
i ,0,22,0 22
The transition dipoles of the eigenstates can be written as a function of those of the local sites
that are the ones that are related to the real structure. We assume that the transition dipoles in the
different helices have the same magnitude:
1,0,0,0 ji
The transition dipoles for the 0+ , 0- and from one quantum states to two quanta states are:
jjii cc ,0,01,0,0 ˆˆ
jjii cc ,0,01,0,0 ˆˆ
jSijiSjjiSijjSiiS cccccccc ,02,021,0, ˆ2ˆ2
jSijiSjjiSijjSiiS cccccccc ,02,021,0, ˆ2ˆ2
jAijiAjjiAijjAiiA cccccccc ,02,021,0, ˆ2ˆ2
jSijiSjjiSijjSiiS cccccccc ,02,021,0, ˆ2ˆ2
jSijiSjjiSijjSiiS cccccccc ,02,021,0, ˆ2ˆ2
jAijiAjjiAijjAiiA cccccccc ,02,021,0, ˆ2ˆ2
The pathways involved in our experiments (19) for T=300 fs can be written in terms of the
eigenstates in the following way:
10
The equivalent pathways starting with a coherence of the asymmetric mode also exist. Assuming
that the experiment involves two distinct timescales the responses can be expressed as an
inhomogeneous average of the homogeneous components. We think this is a good approximation
as we do not identify significant changes in the spectra during the waiting time T. The
orientational prefactors weigh each pathway with the projection of the dipole direction onto the
incident field. Each energy combination will produce a different orientational prefactor which is
then averaged with the inhomogeneous distribution. In the following equations the triangular
brackets represent the inhomogeneous frequency average. Off diagonal disorder is neglected. All
the experiments were done with xxxx polarization. Assuming that the molecules are fixed during
the experiment and that the distribution of angles between the transition dipoles is narrow we
write the response functions in the following way:
11
Positive peaks (indicated in red)
0,0,,0,0
4
,0
21
3Re
15
1,,,,
1
t
TT
ttii
eTRTR
0,0,0,0,
4
,0
54
3Re
15
1,,,,
1
t
TT
ttii
eTRTR
0,0,,0,0,0,0
2
,0
2
,0
13
ˆˆ21Re
15
1,,
1
t
TT
tii
eTR
0,0,0,0,,0,0
2
,0
2
,0
14
ˆˆ21Re
15
1,,
1
t
TT
tii
eTR
0,0,,0,0,0,0
2
,0
2
,0
21
ˆˆ21Re
15
1,,
1
t
TTT
tii
eeTR
0,0,0,0,
,0,0
2
,0
2
,0
25
ˆˆ21Re
15
1,,
1
t
TTT
tii
eeTR
Negative peaks (indicated in blue):
,,,0,0
2
,,0
2
,
2
,0
3
ˆˆ21Re
15
1,,
1
AtA
AA
TT
tAii
eTR
,,,0,0
2
,,0
22
0
3
ˆˆ21Re
15
1,,
1
StS
SS
TT
tSii
eTR
,,,0,0
2
,,0
22
0
3
ˆˆ21Re
15
1,,
1
StS
SS
TT
tSii
eTR
,,0,0,
2
,,0
2
,
2
,0
6
ˆˆ21Re
15
1,,
1
AtA
AA
TT
tAii
eTR
,,0,0,
2
,,0
22
0
6
ˆˆ21Re
15
1,,
1
StS
SS
TT
tSii
eTR
12
,,0,0,
2
,,0
22
0
6
ˆˆ21Re
15
1,,
1
StS
SS
TT
tSii
eTR
,,,0,0
,,0,,0,,0,,0,,,0,0,,,0,0
23
ˆˆˆˆˆˆˆˆˆˆˆˆRe
15
1,,
1
AtA
AAAAAAAA
TTT
tAii
eeTR
,,,0,0
,,0,,0,,0,,0,,,0,0,,,0,0
23
ˆˆˆˆˆˆˆˆˆˆˆˆRe
15
1,,
1
StS
SSSSSSSS
TTT
tSii
eeTR
,,,0,0
,,0,,0,,0,,0,,,0,0,,,0,0
23
ˆˆˆˆˆˆˆˆˆˆˆˆRe
15
1,,
1
StS
SSSSSSSS
TTT
tSii
eeTR
,,0,0,
,,0,,0,,0,,0,,,0,0,,,0,0
27
ˆˆˆˆˆˆˆˆˆˆˆˆRe
15
1,,
1
AtA
AAAAAAAA
TTT
tAii
eeTR
,,0,0,
,,0,,0,,0,,0,,,0,0,,,0,0
27
ˆˆˆˆˆˆˆˆˆˆˆˆRe
15
1,,
1
StS
SSSSSSSS
TTT
tSii
eeTR
,,0,0,
,,0,,0,,0,,0,,,0,0,,,0,0
27
ˆˆˆˆˆˆˆˆˆˆˆˆRe
15
1,,
1
StS
SSSSSSSS
TTT
tSii
eeTR
The averaging due to the inhomogeneous distribution of energies is
ji
jjii
j
jj
i
ii
eii
eTR
jit
TT
jit
2
2
2
2
21221
1
20000
2
0
2
0
1
12
13Re
15
1,,
For the case of uncorrelated frequencies, 0 , equal distribution of energies on both sites and
d being the diagonal trace displaced by c from the center ( dt c ):
2
2
2
2
122
0000
2
0
2
0
12
13Re
15
1,
j
jj
i
ii
ecii
eTR
jidd
TT
jid
13
This same evaluation is done for the rest of the pathways. The FTIR spectrum involves one
quantum transitions only:
2
2
2
2
22
00
2
0
00
2
0
2
1Re
3
1 jjj
i
ii
eii
Sji
jiFTIR
It should be noted that the spectra only depend on the product of coupling constant and cosine of
the angle between the dipoles. This fact limits the information obtained from the spectra to the
absolute value of the coupling constant. Another detail to be observed is that, within our
approximations, decreases with delocalization reaching a lower limit of one half for the
completely delocalized case. The static frequency correlation component () is also distributed
between the symmetric and asymmetric modes reaching a lower limit of 21 when the coupling
is much greater than the inhomogeneous width.
The width of the FTIR and the diagonal trace of the 2D-IR spectra have a quadratic dependence
with the coupling constant. This dependence presents a higher slope in the 2D-IR diagonal trace
which also resolves the symmetric and asymmetric transitions at lower couplings when the
transition is mostly homogeneous. The frequency separation between symmetric and asymmetric
peaks in dimers with disorder is larger than two times the coupling constant when the
inhomogeneous width is comparable to the coupling. This indicates that when static disorder is
present fitting coupled bands with two components and extracting the coupling constant from the
separation between them can lead to overestimations.
The difference in phase between the FTIR spectrum and the 2D-IR diagonal trace widths in 10%
13C=
18O labeled samples shown in Fig. 3 of the main text is attributed to the variation of
homogeneous and inhomogeneous components. When the lineshape is dominated by the
inhomogeneous average shown in the model, there is no significant difference between the 2D-
IR and FTIR lineshapes, whereas if it is dominated by the homogeneous component, the 2DIR
trace is 12 narrower than the FTIR. This makes the phases of the 2D-IR and FTIR i+4
patterns to be different.
14
The diagonal 2D-IR trace presents significant advantages over the FTIR spectra. It is an
inhomogeneous average of a product of Lorenztians whereas the FTIR spectrum consists of the
average of a single Lorenztian. Therefore the 2D-IR diagonal trace has significantly better
spectral resolution than FTIR when the homogeneous broadening is dominant. This fact provides
the 2D-IR diagonal trace with better sensitivity to coupling. To relate to a distribution of
structures, knowledge of the effect of structures on the frequency would be needed (32).
5. Fit of the FTIR and 2D-IR spectra
The model described above was used to fit the FTIR and 2D-IR diagonal traces of 100% and
10% 13
C=18
O labeled samples and extract values for the absolute value of the coupling constant
for each pair of adjacent residues. Demanding consistency between the FTIR and the 2D-IR
spectra provides robustness to the underlying theoretical description because they present
different functionalities of the same parameters. In particular, it constrains the homogeneous to
inhomogeneous ratio by properly describing the relative widths of FTIR and 2D-IR spectra
simultaneously. Also, the uncertainties in the coupling constant (see text) are increased by ca.
20% by dropping consistency between the FTIR and 2D-IR. The results are presented in Fig S7.
15
16
Figure S7: FTIR and 2D-IR diagonal trace fits. Lineshapes of (column 1) normalized FTIR 100%, (column 2)
normalized FTIR 10%, (column 3) normalized 2D-IR 100% and (column 4) normalized 2D-IR 10% 13
C=18
O labeled
samples. The experimental data is shown with full black lines and the fits to the model explained above are shown in
blue dashed lines. Some samples (e.g. G12) exhibit background transitions that are more evident in the 10% 13
C=18
O
labeled samples than in the 100% ones, because the signal to background ratio is reduced. These transitions are
believed to be Fermi resonances to combination modes. In the cases in which any of these peaks would interfere
with the transition under study they were fitted with a Gaussian (green) while the transition of interest was fitted
with the model developed above (blue) resulting in the total spectrum (red).
17
6. Structure determination:
In order to create a complete family of possible two-fold symmetric ideal helical dimers the
procedure shown in Fig. S8 was used.
Fig. S8: Sampling the of two-fold symmetric ideal
helix dimers space. The axes of the two fold
symmetric ideal helices lies along the z axis. The
phase around the z-axis was varied between -180 and
180 degrees in steps of 10 degrees which is equivalent
to approximately a 0.5 Å displacement on the surface
of the helix. The translation along the z axis leaves
the crossing point of the helix on the xy plane. This
displacement was sampled in a range of 20 Å in steps
of 0.5 Å. The helices were rotated around the x axis
by the crossing angle divided by 2 which was
sampled between -180 and 180 degrees in steps of 5
degrees. Finally, each helix was displaced in opposite
directions along the x axis by half the interhelical distance which was sample from 6 Å to 10 Å in steps of 0.25 Å.
For each structure the vibrational coupling constant was calculated assuming that the dipole
direction of the 13
C=18
O substituted dipole is the same as the unperturbed 12
C=16
O one. Through
perturbation theory the angle between unperturbed ( m ) and perturbed ( 'm ) transition dipoles
can be calculated as:
n
mnmn
m
mmm
cos
65
11cos
'
'
where mn is the coupling in cm-1
, mn is the angle between the m and n transition dipoles and the
energy gap is 65 cm-1
. For central residues in an alpha helix these angles are smaller than 3.
These yielded a family of 1.9 x 106 structures 48 of which possessed vibrational coupling
constants whose absolute value agreed with the experiment by a 2 analysis with 75%
confidence. This group of structures had a crossing angle of -63±13 and interhelical distance of
8.5±0.3 Å.
A new module named IR has been implemented into the Xplor-NIH (25,26) program in order to
use IR restraints in protein structure calculation. The structure was calculated by simulated
annealing in torsion angle space using experimental IR constraints together with backbone
dihedral angle constraints and hydrogen bonds for ideal -helical secondary structure elements.
18
The 48 preselected structures obtained by the method described above were each taken as
starting points of simulated anneal runs for 100 structures. Every structure was first minimized
by 500 steps to remove bad contacts, bathed at a high temperature (3000 K) for 1000 steps,
followed by cooling to a low temperature (10 K) for another 1000 steps and subjected to Powell
minimization for final 2000 steps. We found the force constants for the IR constraints (in cm-1
)
by reducing a coarse sampling (5-100 Kcal mol-1
cm2) to a smaller range (10 to 40 kcal mol
-
1cm
2), in which the structural RSMD values converged and the overall secondary structure was
not distorted (Table 1).
Table S1: Refinement statistics for protein structures in presence of IR constraints with different weight factors
Weight factor 10
kcal mol-1
cm2
20*
kcal mol-1
cm2
40
kcal mol-1
cm2
Structure statistics
Average RMSD. to the mean structure (Å)
Residue 10-21 0.92 ± 0.23 0.78 ± 0.25 0.85 ± 0.11
All residues 1.28 ± 0.20 1.14 ± 0.21 1.16 ± 0.09
Fitting
Slope 0.95 ± 0.06 0.97 ± 0.05 0.97 ± 0.05
R 0.968 0.973 0.971
RMS (cm-1
) 0.713 0.693 0.705
Energy (kcal)
Total energy 35.7 ± 1.3 38.5 ± 2.5 43.6 ± 4.8
IR energy 0.6 ± 0.4 1.1 ± 0.6 2.3 ± 1.7
*: the one was used in the final calculation.
Importantly, the RMSD between the mean structures computed with force constants between 10
and 40 kcal/mol is smaller than the RMSD computed for the individual members of the ensemble
at a given value of the force constant. This can be understood, because the structural restraints
19
maintain helical geometry and prevent repulsive interactions between the helices, while the
experimental restraints provide the bulk of the attractive potential. The final value of the force
constant in the simulated annealing target function for IR (20 kcal mol-1
cm2) was chosen based
on the smallest RMSD and the best agreement between the experimental coupling constants with
those back calculated from the structure (Table S2 and Fig. S9).
Table S2. Pairwise RMSD. (Å) between the mean structures obtained with different weights for IR constraints.
10
kcal mol-1
cm2
20*
kcal mol-1
cm2
40
kcal mol-1
cm2
10
kcal mol-1
cm2
0 0.20 0.38
20*
kcal mol-1
cm2
0 0.42
40
kcal mol-1
cm2
0
Figure S9: Pairwise backbone RMSD, calculated for 20
best structures by superimposing residues 10-21. Magenta,
red and purple traces represent values with an IR-restrained
force constant of 10, 20 and 40 kcal mol-1
cm2, respectively.
Although the convergence is best for N- and C-terminus
with an IR-restrained force constant of 40 kcal mol-1
cm2,
the central part where the IR constraints are actually
applied has the best converge for 20 kcal mol-1
cm2.
The final values for the IR constraint is 20 kcal mol-1
cm2; the rest of the restraints were standard
(50 kcal mol-1 Å-2
for hydrogen bond restraints; 5 kcal mol-1
rad-2
for dihedral angle restraints; 4
kcal mol-1 Å-4
for the quartic van der Waals repulsion term). Of the 4800 obtained structures, the
20 lowest energy structures form an ensemble to represent the structure. The distribution of
20
crossing angles and interhelical distances and the RMSD. per residue is presented in Fig. S10.
Figure S10: Simulated
annealing results: (left)
distribution of crossing
angles and interhelical
distances for the 20
structures yielded by the
constrained simulated
annealing. The red dot
belongs to the structure of
lowest energy. (right) RMSD
per residue for the 20 yielded
structures.
References and Notes 1. P. Hamm, M. Lim, R. M. Hochstrasser, Structure of the amide I band of peptides measured by
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