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: hochstra@sas.upenn.edu

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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