Supporting Information

Site-Specific Hydrogen Exchange in a Membrane

Environment Analyzed by Infrared Spectroscopy

Esther S. Brielle∗ and Isaiah T. Arkin∗

The Alexander Silberman Institute of Life Sciences. Department of Biological Chemistry.

The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem, 91904, Israel.

E-mail: esther.brielle@mail.huji.ac.il; arkin@huji.ac.il

1

H/D exchange in a bifurcated system

In order to further examine the impact of H/D exchange on the amide I vibrational mode, we

analyzed a bifurcated hydrogen bonding system. Such systems are common in proteins,1,2

and are particularly prevalent in helices containing serine, threonine and cysteine residues.3,4

In particular, we have previously shown that the carbonyl group of Phe26 in the SARS E

protein is hydrogen bonded to two protons: the amide proton and the side-chain hydroxylic

H+ of residue Thr30.5 Furthermore, the bonding of two protons to the single carbonyl results

in a 60% increase in the bond strength, and a -15 cm−1 shift of the amide I vibration.5

Considering the above, we collected the FTIR spectra for two naturally found variants

of the SARS E protein: Ser30 that supports bifurcated hydrogen bond formation to Phe26,

and Val30 that does not. Comparisons between the two would enable us to determine how

the presence of a bifurcated hydrogen bond affects the 1-13C=18O amide I band upon H/D

exchange.

The results shown in Supporting Fig. 1 indicate that the carbonyl that is hydrogen

bonded to two hydrogens does not have an increased H/D band shift in comparison to the

non-bifurcated system (i.e. canonical hydrogen bond). If the H/D exchange of the hydrogen

bonding hydrogens influences the amide I frequency shifts, one would expect that stronger

bonds would result in larger shifts. However, deuteration of the bifurcated system (Ser30)

results in a smaller shift in comparison to the single, canonical hydrogen bond (Val30).

These results prove that the amide I shift arising from H/D exchange is not due to

the hydrogen bonding hydrogen (or hydrogens in the case of a bifurcating system), but

rather solely due to the covalently bonded amide hydrogen. Hence, the variation in the shift

magnitudes upon H/D exchange, as seen in Fig. 2 of the main manuscript, arises due to

differences in the micro-environment of the amide hydrogens.

2

1570158015901600 156016100

0.2

0.4

0.6

0.8

1.0

Abso

rban

ce (a

.u.)

Wavenumber / cm-1

Val30

Phe26

Phe26

Ser308.5 cm-1

10 cm-1 Val30

Phe26

13CCH3N

H

O18

NH

HCHC

H3C

H3C

C

O

13CN

O18

NHC

CH2

O

C

O

D

DD

13CND

O18

ND

HCHC

H3C

H3C

C

O

Phe26

Ser30

13CNH

O18

NH

HCCH2

O

C

O

H

H/D exchange

H/D exchange

0

0.2

0.4

0.6

0.8

1.0

Supporting Figure 1: FTIR spectra in the isotope edited amide I mode region of two SARSE peptides: Val30 (top) and Ser30 (bottom). In both peptides the 1-13C=18O label is atposition Phe26. Spectra of protiated systems are in black, while deuterated samples areshown in red. The respective hydrogen bonding schemes of the two systems are depicted onthe right, utilizing the coloring detailed in Fig. 3 of the main manuscript.

3

Time average electrostatics in a structural context

The results depicted in Supporting Fig. 2, show the localized electrostatics across the different

sites. These electrostatic values were used to calculate the correlation seen in Fig. 4 of the

main manuscript to the extent of the hydrogen exchange impact on the amide I vibrational

mode.

Experimental

Peptide synthesis

The peptides that were used for the experimental procedures correspond to residues Ser22 to

Leu46 of the influenza A M2 H+ channel: SSDPLVVAASIIGILHLILWILDRL; and residues

Glu7 to Arg38 of the SARS E protein: EETGTLIVNSVLLFLAFVVFLLVTLAILTALR. In

both instances, the peptides encompass the tetrameric (influenza A M2 H+ channel) or

pentameric (SARS E protein) membrane spanning domain, and exhibit most of the func-

tionalities of the full length proteins.9–11

Five different peptides were synthesized for each protein, each with a single 1-13C=18O la-

bel at different positions, corresponding to the residues listed in Fig. 2 of the main manuscript.

The peptides were synthesized using solid-phase N-(9-fluorenyl methoxycarbonyl) chemistry,

where the specific labeled amino acids were introduced as precursor amino acids during the

relevant step in synthesis. The 1-13C=18O labeled amino acid preparation is described pre-

viously.12,13

Peptide purification

The ten peptides were purified by dissolving ca. 2 mg of crude peptide in 2 ml of trifluoroacetic

acid and then injecting it into a 20 ml Jupiter 5 C4 300 A reverse phase high performance

liquid chromatography column (Phenomex, CA). The column was pre-equilibrated with 80%

4

Val27

Val28

Ala29Ala30

Leu40

15Electrostatics (kT/e)5

Supporting Figure 2: Representation of the influenza A M2 tetramer simulation frame along-side a depiction of the channel pore as determined by HOLE.6 The different amide carbonylpositions (of residue i indicated in the figure) that we examined by FTIR are shown in yellowlicorice representations. The amine groups (of residue i + 1) of the peptide bond are alsodepicted in licorice representations, but their color value is determined by their time-averageelectrostatics calculated using the Particle mesh Ewald method7 as implemented in vmd.8

5

H2O, 8% (w/v) acetonitrile and 12% (v/v) isopropanol. The peptide was introduced in the

column at these pre-equilibrated solvent proportions and then eluted by linearly decreasing

H2O to 0% while linearly increasing acetonitrile and isopropanol proportions, while main-

taining a 2:3 ratio between them. The final solvent composition reached 40% acetonitrile

and 60% isopropanol. All solvent solutions contained 0.1% (v/v) trifluoroacetic acid.

H/D exchange and peptide reconstitution in membranes

The peptide containing fractions were divided into two identical aliquots, in order to make

two samples. In the first sample all exchangeable hydrogens were deuteriums, while in the

latter, they were all protiums.

To prepare the deuterated sample, 15µl of concentrated DCl was added to the eluted

fractions, followed by freezing with liquid nitrogen and lyophilization. Subsequently, 0.5 mg

of peptide was dissolved in 640µl 2,2,2-Trifluoro-ethanol-d3 (TFEd3) and then 15µl of deu-

terium chloride solution 30% was introduced to ensure an acidic environment in order to

facilitate H/D exchange. After 3 hours of incubation and mixing at 37◦C, the sample was

combined with 2.5 mg of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, which was also dis-

solved in 25µl of TFEd3. The sample was further incubated and mixed at 37◦C for another

hour and then desiccated overnight in order to remove any TFEd3. 1 ml of deuterium oxide

was added to the sample to create liposomes containing the purified peptide in a completely

deuterated solution.

The protiated sample was prepared in an identical manner, whereby all deuterated sol-

vents were replaced with their protiated equivalents. Final peptide samples contained ca.

0.5 mg/ml protein and 2.5 mg/ml lipid.

FTIR spectra collection

Attenuated total reflection (ATR) FTIR spectra were collected using a Nicolet Magna 560

spectrometer (Madison, WI), equipped with a high-sensitivity liquid nitrogen-cooled MCT/A

6

detector. Approximately 200µL of each sample were deposited on a trapezoidal Ge internal

reflection element (50 × 2 × 20 mm), which is part of a 25-reflection ATR accessory from

Graseby Specac (Kent, England). The sample was purged inside the spectrophotometer with

water- and CO2-depleted air. 1000 interferograms at 0.5 cm−1 resolution were processed with

1-point zero filling and Happ-Genzel apodization. The 1000 measurements were collected

in this way and averaged to produce each FTIR spectrum using OMNIC software. OMNIC

software automatic smoothing and automatic baseline correction were applied to each of the

spectra in Fig. 2 of the main manuscript. The H/D shifts in Fig. 4b of the main manuscript

(x-axis values of the graph) were determined as the difference between the peak values of D

spectra and H spectra. For shoulder D spectra that did not have a distinct peak position, (M2

A29 and V28) the second derivative was used to determine peak position. The x error bars

were calculated as the uncertainty in peak positions, averaged over all the spectra collected

for that position. The minimum error is the resolution of the FTIR instrumentation.

DFT simulations

All DFT optimizations and frequency calculations were performed using Q-Chem software14

with the aug-cc-pvdz basis sets15 and an EDF2 exchange-correlation functional.16 The sol-

vent dielectric was set to 4 to mimic the apolar membrane environment. Geometry optimiza-

tion was performed on the first model system in Fig. 3 of the main manuscript, to mimic

canonical i to i+4 hydrogen bonds found in α-helices. Non-hydrogen atoms were constrained

during geometrical optimization to retain the correct helical structure. Following geometric

optimization, frequency calculations were performed for all 4 model systems seen in Fig. 3

of the main manuscript, with both 1-13C=18O and H/D isotope labeling.

MD simulations set up

The current study involves MD simulations of the influenza A M2 H+ channel. The initial

structure is derived from PDB code 4QKM, an X-ray diffraction protein structure in a

7

membrane-like environment.17

The simulation parameters in GROMACS version 5.0.118 that were used were the GRO-

MOS96 53a6 force field19 and a simple point charge (SPC) water model.20 The lipid bilayer

force field parameters are based on those derived by Berger et. al.21 The lipid bilayer used in

the simulation was a pdb structure file and topology file of 1-palmitoyl-2-oleoyl-sn-glycero-

3-phosphocholine (POPC), provided by the Tieleman group.22

Our initial protein structure, the 4QKM tetramer was placed in the POPC membrane in a

manner such that the protein pore axis coincided with the membrane normal and the protein

stretched in this direction across the hydrophobic membrane bilayer. All lipid molecules that

were found to have any atom within 1A of a protein atom were removed from the system.

Three of the four His37 side chains were protonated as was shown characteristic of the open

M2 channel.23 The charge of the system was neutralized and the salt concentration was

raised to 140 mM by adding 23 Na+ and 22 Cl− ions. Upon completion, the simulation

system contained 94 lipid molecules and 2368 water molecules along with the ions and the

four transmembrane peptide chains.

MD simulation process

Initial energy minimization was attained by a process of several steps. The system was

first allowed to converge to Fmax < 1000 kJ/(mol · nm) with the steepest descent algorithm.

This was followed by allowing the system to converge to Fmax < 100 kJ/(mol · nm) using

the conjugate gradient algorithm. Finally, the system was allowed to converge to machine

precision using the conjugate gradient algorithm.

This energy minimization was followed by a series of three positional restraint simulations

of 1 ns in length with a time step of 2 fs in which the protein was held under harmonic

positional restraints with spring constants of k = 1000 kJ/(mol · nm2), followed by k =

500 kJ/(mol · nm2), and lastly k = 0 kJ/(mol · nm2). This series of positional restraint

simulations allows for a tighter packing of lipid molecules around the positionally restrained

8

protein.

The positional restraint simulations were then followed by molecular dynamic simulations.

The system was first allowed to equilibrate for 1ns and then a 10ns production simulation

was performed, each with a 2 fs time step. The final production simulation atom coordinates

were saved every 10ps and provided the positional and electrostatic information from which

the in-plane electric field acting on the -NH hydrogen was calculated (y-axis values of Fig.

4b of the main manuscript ).

Throughout all of the MD processes, all covalent bond lengths and angles were con-

strained using the LINCS algorithm.24 A Nose-Hoover temperature thermostat25,26 main-

tained a relatively constant temperature of 310K (37◦C) in separate constant temperature

baths for the protein, lipid and water+ion groups with a temperature coupling time constant

of τ = 0.5ps for each group. The semi-isotropic Parrinello-Rahman barostat27,28 maintained

a relatively constant pressure of 1 bar for the entire system with a pressure coupling time

constant of τ = 2ps. Van der Waals interactions and Coulombic interactions were both cut

off at 1.2nm. The Particle mesh Ewald (PME) method7 was used to calculate long-range

electrostatic interactions that extended beyond this 1.2nm cutoff.

Electrostatics and electric field calculations

The production simulation output trajectory file was visualized in Visual Molecular Dynam-

ics (VMD).8 In order to determine the electric field contribution to the NH in-plane bending

motion, the (x,y,z) position was calculated for each C, N, and H amide atom for each helix

at each of the pertinent sites in VMD for each frame in the production simulation trajectory

(for each frame, 60 positions were recorded: 4 C positions for Val27, 4 N and H positions

for Val28; 4 C positions for Val28, 4 N and H positions for Ala29; 4 C positions for Ala29,

4 N and H positions for Ala30; 4 C positions for Ala30, 4 N and H positions for Ser31; 4

C positions for Leu40, 4 N and H positions for Trp41). The NH stretch motion direction is

parallel to the−−→NH vector (see Fig. 4a of the main manuscript). The NH out-of-plane bend-

9

ing motion is perpendicular to both the−−→CN and the

−−→NH vectors, and so was determined as

the cross product between the two. The NH in-plane bending motion is perpendicular to the

−−→NH and the out-of-plane bending vectors and so was determined as the cross product of the

two. In this way, the in-plane bending motion vector can be determined for each relevant

amino acid H site (28, 29, 30, 31, 41) for each chain for each frame.

The PMEPot plugin29 was used to calculate the potential in a voxel grid pattern over

the production simulation space for each frame. The electric field (E) was calculated from

the potential voxel grid (V ) as the negative gradient, E = −∇V , for each voxel grid position

in each frame. The E for each relevant H position was projected onto the in-plane bending

motion unit vector to achieve only the relevant Ein−plane bend. The Ein−plane bend values of

each frame and helix were averaged in order to receive the y-value depicted in Fig. 4b of the

main manuscript for each NH position. The y error bars were determined per helix as the

standard deviation of each value divided by√n, where n is the number of values averaged.

Then the different helix errors for each NH position were averaged.

Acknowledgement

This work was supported in part by grants from the binational science foundation (2013618)

and the Israeli science foundation (175/13).

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