Supporting Information
Wiley-VCH 2014
69451 Weinheim, Germany
Irreversible Denaturation of Proteins through Aluminum-Induced
Formation of Backbone Ring Structures**
Bo Song,* Qian Sun, Haikuo Li, Baosheng Ge, Ji Sheng Pan, Andrew Thye Shen Wee,
Yong Zhang, Shaohua Huang, Ruhong Zhou, Xingyu Gao,* Fang Huang,* and Haiping Fang
anie_201307955_sm_miscellaneous_information.pdf
1. DETAILS OF APPLIED METHODS
1.1 Calculations using ab initio methods. The calculations of the short peptide HCO-Ala-NH2
(C4N2O2H8) were performed using an ab initio method based on the second-order Møller-Plesset
perturbation theory (MP2).[23] To realize the relaxation on the long peptide HCO-[Ala]5-NH2, we applied
an ab initio method based on the density functional theory (DFT), where the B3LYP functional[s1,s2]
within the generalized gradient approximation (GGA) in the framework of DFT was used. The 6311++G(d,p) basis set of triple-Zeta quality and including diffuse functions was applied on all atoms. The
calculations were carried out using the Gaussian-09 package.[s3]
The initial state in the calculations was: the peptide HCO-Ala-NH2 (C4N2O2H8) and a hydrated Al ion
[AlOH(H2O)4]2+ separated by a far distance (Fig. 1a). Here, the five-coordinate hydrated Al ion with two
positive charges was applied for the biochemically critical pH range of 4.3 to 7.0.[22]
(I)
The binding energy Ebinding
( Al) of the Al ion bound with the oxygen in the backbone of State I (Fig.
1b) was calculated via the following formula:
(I)
E binding
( Al ) = E ([C 4 N 2 O 2 H 8 AlOH(H 2 O) 3 ]2 + ) + E ( H 2 O)
− E (C 4 N 2 O 2 H 8 ) − E ([ AlOH(H 2 O) 4 ]2 + ),
where E([C4N2O2H8AlOH(H2O)3]2+) denotes the energy of the peptide with the hydrated Al ion bound in
State I, E(H2O) indicates the energy of a water molecule, while E(C4N2O2H8) stands for the energy of the
pristine peptide, and E([AlOH(H2O)42+]) for the energy of the pristine hydrated Al ion.
(I)
The binding energy Ebinding
(M ) (M = Na, K, Mg or Ca) of the M ion bound with the oxygen in the
backbone of State I was calculated via the following formula:
(I)
Ebinding
(M ) = E ([C 4 N 2O 2 H8 M(H 2O)5 ]n + ) + E (H 2O)
− E (C 4 N 2O 2 H8 ) − E ([M(H 2O)6 ]n + ),
where n = 1 when M = Na or K, and n = 2 when M = Mg or Ca. E([C4N2O2H8M(H2O)5]n+) denotes the
energy of the peptide bound with the hydrated M ion in State I, and E([M(H2O)6]n+) indicates the energy
of the hydrated M ion.
(II)
( Al) for State II (Fig. 1c) of the hydrated Al ion simultaneously binding
The binding energy E binding
to the atoms N1 and O2 in the peptide backbone was calculated via the following formula:
(II)
Ebinding
(Al) = E ([C 4 N 2 O 2 H 7 AlOH(H 2 O) 2 ]+ ) + E ([H 3O]+ ) + E ([H 2 O])
− E (C 4 N 2 O 2 H 8 ) − E ([AlOH(H 2 O) 4 ]2+ ),
where E([C4N2O2H7AlOH(H2O)2]+) denotes the energy of the peptide with the hydrated Al ion bound in
State II, while E([H3O]+) indicates the energy of a hydronium ion consisting of the dropped hydrogen ion
(H1) from the peptide and the dropped water molecule from the hydrated Al ion.
(II)
(M) for State II of the M ion in the hydrated group simultaneously
The binding energy Ebinding
binding to the atoms N1 and O2 in the peptide backbone was calculated via the following formula:
(II)
Ebinding
(M ) = E ([C 4 N 2O 2 H 7 M(H 2O) 4 ]( n−1)+ ) + E ([H 3O]+ ) + E ([H 2O])
− E (C 4 N 2O 2 H 8 ) − E ([M(H 2O)6 ]n+ ).
E([C4N2O2H7M(H2O)4](n-1)+) denotes the energy of the peptide with the hydrated M ion bound in State II.
1.2 Classical molecular dynamics simulations. We performed classical dynamics simulations using one
poly-Ala peptide [Ala]5 in AlCl3 solution. The concentration of the Al ions was set at 50 mM. The
simulations were carried out at a constant pressure of 1 bar, a temperature of 350 K and an initial box size
of 5.0 nm × 5.0 nm × 5.0 nm with Gromacs 4.0,[s4] using the particle mesh Ewald method[s5] for full
electrostatics with a cut-off of 1 nm. Considering that an Al ion in water usually binds to one hydroxyl
group under the biochemically critical pH range of 4.3 to 7.0,[22] we applied a form of [Al-OH]2+. The
Amber03 force field[s6] was used for the peptide, the Cl ions of AlCl3 in the solution, and the hydroxyl
group of [Al-OH]2+. In order to maintain the +2 e charge of the [Al-OH]2+, based on the NBO analysis,
we modeled the Al ion as the particle with a charge of qAl = +2.45 e. For the Lennard-Jones (L-J)
potential of the Al ion, the cross-section and depth were set as σAl = 0.40 nm and εAl = 0.5045 kcal/mol,
respectively.[s7] An Al-O bond length of 0.17 nm and Al-O-H bond angle of 128° were maintained by
harmonic potentials with spring constants of 68,665 kcal/mol·nm-2 and 44 kcal/mol·rad-2 before
relaxation, where the parameters were calculated based on the MP2 method. The TIP3P water model[s8,s9]
was applied. A time step of 1 femtosecond (fs) was used, and data were collected every 1 picosecond (ps).
1.3 Circular dichroism spectra. Denaturation experiments were performed using several salts,
including Al2(SO4)3, K2SO4, MgSO4 and CaSO4. Due to the poor solubility of Al3+ at neutral pH, the
experiments were carried out at pH = 3.8. The final concentration of Al2(SO4)3 was 1 mM, while the
concentrations of the other salts reached 2 mM. In all experiments, the N-terminal domain of PGK was
dissolved in sodium acetate buffer (200 mM sodium acetate plus the corresponding salt, pH = 3.8) to keep
the solution stable when Al2(SO4)3 was added. The concentration of the N-terminal domain of PGK was
30 µM. The denaturation experiments were carried out in the temperature between 278 and 368 K.
1.4 X-ray photoelectron spectra. For X-ray photoelectron experiments, the buffer solution, the protein
concentration, and the preparation of irreversibly thermodenatured samples were the same as in the
above-described circular dichroism experiments. Sample 1 was initially mixed with 1 mM Al2(SO4)3, and
Sample 2 with both 1 mM Al2(SO4)3 and 2 mM MgSO4. After being irreversibly denatured and
aggregated, the samples were transferred into centrifugal tubes with the bottom covered by a copper sheet,
then centrifuged in horizontal centrifuge rotors at 18,000 × g for 30 minutes. The supernatants were
discarded and the samples were washed twice with ultrapure water. In this way, the aggregated proteins
depositing on the copper substrate were prepared as the samples for XPS experiments. The XPS
experiments were performed using a VG ESCALAB 220i-XL instrument equipped with a monochromatic
Al Ka (1486.7 eV photons), a concentric hemispherical analyser, and a magnetic immersion lens (XL
lens) to increase the sensitivity of the instrument. The background pressure in the chamber was in the low
10−10 mbar range during analysis. The instrument was calibrated with pure gold. All spectra were
recorded in the constant pass energy mode of the analyzer using the monochromatic Al Ka X-ray source.
Survey spectra were recorded with a pass energy of 150 eV and a 1-eV step width. The narrow scans for
high-resolution spectra were recorded with a pass energy of 20 eV and 0.1-eV step width. The spectra in
Fig. 3 show that the Al 2s peak can be distinguished from the Cu 3s peak.
1.5 Calculations of Al-2s binding energy. Using an ab initio method based on the relativistic density
functional theory, we calculated the binding energy of a core electron (CE) on the 2s level of Al (Al-2s)
in the Al-involved molecule. To include the relativistic effects of the core electrons, we applied the exact
two component method (X2C).[s10,s11] The P86 exchange[s12] and P86 correlation[s13] functional of the
generalized gradient approximation was employed with self-interaction correction (SIC) according to
Stoll et al[s14]. The STO-TZ2P basis set of triple-Zeta quality was applied on all atoms. The calculations
were carried out with the BDF program package.[31]
The binding energy of core electron on the 2s level of Al (Al-2s) in the Al-involved molecule was
CE
( Al - 2 s ) = E ( Al - 2 s1) − E ( Al - 2 s 2) .[s15] Here, E(Al-2s2) denotes the
calculated by the formula Ebinding
energy of the neutral molecule that the Al-2s level is fully occupied with two electrons, E(Al-2s1)
indicates the energy of the core-ionized molecule that the Al-2s level is half occupied with one electron.
2. FURTHER RESULTS AND DISCUSSIONS
2.1 Simulations and discussion of the Al ions contacting the O atoms on the peptide backbone
Based on our classical molecular dynamics (MD) simulations, to determine the contact of the Al ions
with the O atoms on the peptide backbone (labeled by Opb) in water, the smallest distance d between the
Al ions and the kth Opb atom ( O kpb ) in the peptide was calculated per 10 snapshots. We specified the Al
ion contacting the O kpb atom (i.e., no water molecule between Al and O kpb ) when d < 0.35 nm. In Fig. S1,
there present the results of the smallest distance d. In many intervals of time (such as, t = 23.70 ~ 23.85 ns
for O 2pb , 23.31 ~ 23.44 ns for O 3pb ), the smallest Al - O kpb distances are smaller than 0.35 nm, indicating
the Al ions can conquer the potential barrier in water and contact the O kpb atoms during these times. The
contacts are unstable in the MD simulations, because the charge −0.55 e of the Opb atom is weaker than
the charge −0.80 e of the O atom in a water molecule (labeled by Owm), leading to the interaction of Al
with Opb weaker than the interaction with Owm if only considering the electrical interaction within the
classical force field. However, as suggested by the ab initio calculations in the main text, due to the
charge transfer and the covalent bond between Al and Opb in the peptide, the interaction of Al with Opb is
stronger of 27.05 kcal/mol than the interaction with Owm. Thus, we conclude that the O atoms on the
backbone are able to absorb Al ion stably in the presence of water.
Figure S1. Smallest distance (d) of the Al ions with the kth oxygen atom ( O kpb ) on the backbone of
the poly-Ala peptide [Ala]5 with respect to the simulation time. Ignoring the terminal effect, we just
consider the two oxygen atoms (black curve for k = 2 and red curve for k = 3) in the middle area of the
peptide. The green line denotes the critical distance of 0.35 nm. The snapshot at 24 nm is presented in the
inset. The cyan, blue, red, white and green balls represent carbon, nitrogen, oxygen, hydrogen and
aluminum, respectively. The yellow ribbon represents the peptide backbone, while the dotted lines
indicate the Al-O distance. There is no water molecule between the atoms Al and O 2pb .
2.2 Content of amino acids in the sequence
The sequence of the applied N-terminal domain, containing residues 1 to 174 of the full-length PGK
coded by 1PHP in Protein Data Bank, is as follows:
MNKKTIRDVDVRGKRVFCRVDFNVPMEQGAITDDTRIRAALPTIRYLIEHGAKVILASHLGRPKGKVVEELRLD
AVAKRLGELLERPVAKTNEAVGDEVKAAVDRLNEGDVLLLENVRFYPGEEKNDPELAKAFAELADLYVNDAF
GAAHRAHASTEGIAHYLPAVAGFLMEKE
It is rich in nonpolar and neural amino acids (such as glycine, alanine and valine), poor in negatively
charged amino acids, and much poor in aromatic amino acids (Fig. S2).
Figure S2. Content of amino acids in the N-terminal domain of PGK. In the left, middle and right
highlighted areas, there are the nonpolar-and-neutral, aromatic and negatively-charged amino acids,
respectively.
2.3 Further discussion on XPS results
In X-ray photoelectron spectrum for the denatured PGK protein (Fig. S3), there are C 1s, N 1s,O 1s, Cu
2p3/2 and Cu 2p1/2 photoemission peaks together with three Cu LMM Auger peaks. The peak at about
76 eV (1753 kcal/mol) are mainly from Cu 3p overlapped with photoemission from Al 2p. The small
features at about 120 eV (2767 kcal/mol) are from Al 2s and Cu 3s photoemission, which is well resolved
in the XPS narrow scan as shown in Fig. 3 of the main text.
Figure S3. Survey of X-ray photoelectron spectrum for the irreversibly denatured and aggregated
PGK protein depositing on a copper substrate. (a) The sample of the PGK protein only mixed with
Al ions. (b) The Sample of the PGK protein mixed with Al and Mg ions. The upper scale (green)
shows the binding energy in the unit of kcal/mol. The Cu-related peaks come from the copper substrate
that PGK deposits on.
The Cu 3s photoemission peak with its binding energy comes from the Cu substrate oxidized slightly
on the surface before the sample preparation ex situ, which is also confirmed from the Cu 2p XPS
spectrum.
The Al 2s binding energy of 117.3 eV (2705 kcal/mol) indicates that the aluminum in the sample
does not come from the aluminum sulfate used in the solution, because those binding energies of trivalent
Al ions are with much higher values. For example, Al 2s photoemission peak of aluminum sulfate was
found at a binding energy of about 118.3 eV (2728 kcal/mol).[s16] In fact, the binding energy of 117.3 eV
is more similar to the Al-2s binding energy of 117.8±0.2 eV in metallic aluminum (Fig. S4).
Figure S4. X-ray photoelectron spectrum of oxidized Al metal. The red curve denotes the
experimental results, fitted by the black and blue curves. The left peak at 117.8±0.2 eV falls in the area of
metallic Al 2s. The right peak at 120.8±0.2 eV comes from Al 2s of Al2O3.
2.4 NMR results and discussion
We have performed NMR experiments of an unstructured short peptide (RGDS) by Al2(SO4)3 and
Na2SO4, together with the control experiment without sulfate salt. The concentration of the short peptide
was 1 mM. In order to keep the same effect of sulfate ions, the concentration of Al2(SO4)3 and Na2SO4
was set to 4.5 mM and 13.5 mM, respectively. The experiments were carried out at pH = 3.8 with a
sodium acetate buffer of 200 mM. All NMR experiments were performed on a Bruker AVANCE III 600
MHz spectrometer operating at 600.13 MHz. For all 1H-NMR experiments, the acquisition parameters
were set as follows: Pulprog = p3919 gp, SW = 15 ppm, TD = 32 K, D1 = 2 S, NS = 256, T = 298.0 K,
and the processing parameters: WDW = EM, LB = 0.3 Hz. For TOCSY experiment, the acquisition
parameters were set as follows: Pulprog = mlevgpphprzf, SW1 = SW2 =15 ppm, TD (F2) = 2K, TD (F1)
= 256, D1 = 1.5 S, D9 = 0.1 S, NS = 40, DS = 16, T = 298.0 K, and the processing parameters: TD (F2) =
TD (F1) = 2K, WDW (F2) = WDW (F1) = QSINE, SSB (F2) = SSB (F1) = 2. Additionally, the Sodium
Trimethylsilylpropionate (TSP) was used as an external reference in NMR experiments, and the intensity
of NMR signals of all 1H-NMR spectra was determined by the intensity of TSP signals. To present the
intensity of 1H-NMR signal clearly, the integration of the signal was carried out in the Bruker software
TopSpin 2.1, and calibrated by the integral of the NMR signals of TSP. The NMR-signal integration value
was determined via bias correction.
Fig. S5. NMR spectra of an unstructured peptide RGDS. a) 1H-NMR spectra with Na2SO4 (upper
curve), Al2(SO4)3 (middle curve) without sulfate salt (lower curve). b) 1H-1H TOCSY NMR spectra with
no sulfate salt. c) Molecular structure of the peptide RGDS.
In the case of no sulfate salt, four signals are observed in the region of chemical shift δ = 7.0 ~ 9.0
ppm (Fig. S5a), which fall in the area of H atom binding to amide N. As suggested by 1H-1H TOCSY
NMR spectra (Fig. S5b), the signals S1 ~ S3 correspond to the three H atoms binding to the amide N on
the backbone, and the signal S4 corresponds to the H atom binding to the amide N of the Arg residue (Fig.
S5c). When Na2SO4 is added, as presented by the NMR-signal integration value in Table S1, the intensity
of the four signals S1 ~ S4 simultaneously reduces to about one third of the intensity of the signals in the
case without sulfate salt. This results from the screening effect of sulfate salt on NMR signals.
Interestingly, when Al2(SO4)3 is added, the intensity of the signals S1 ~ S3 (corresponding to the HN on
the backbone) further reduces to about two third of the intensity of the signals in the case of Na2SO4,
while the intensity of the signal S4 corresponding to the HN of the Arg residue is still similar to the
intensity of the signal in the case of Na2SO4 (seeing Table S1). It should be noticed that the Al-ion
concentration (9.0 mM) is only one third of Na-ion concentration (27.0 mM), to keep the same
concentration of sulfate ions (13.5 mM). Therefore the additional reduction of the first-three-signal
intensities can be assigned to the substitution of HN by Al, and the substituted HN atoms cannot be
distinguished from the water signal in the 1H-NMR.
Table S1. Integration value of the 1H-NMR signal of the amide H atom in the peptide RGDS. The
integration value indicates the intensity of the NMR signal. The 1H-NMR signals S1 ~ S3 correspond to
the three amide H atoms on the backbone, and the signal S4 corresponds to the one amide H atom of the
Arg residue.
S1
S2
S3
S4
No sulfate salt
0.70
0.78
0.80
0.65
Na2SO4
0.27
0.28
0.30
0.22
Al2(SO4)3
0.19
0.19
0.21
0.21
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