Monitoring of Zwitterionic Proline and Alanine Conformational Space by Raman Optical Activity
description
Transcript of Monitoring of Zwitterionic Proline and Alanine Conformational Space by Raman Optical Activity
Monitoring of Zwitterionic Proline and Alanine Conformational Space by Raman Optical Activity
Josef Kapitán a,b, Petr Bouř b and Vladimír Baumruk a
a Institute of Physics, Charles University, Ke Karlovu 5, Prague, 12116, Czech Republicb Institute of Organic Chemistry and Biochemistry, Flemingovo nám. 2, Prague, 16610, Czech Republic
REFERENCES:
[1] L.D. Barron, L. Hecht, E.W. Blanch, A.F. Bell, Prog. Biophys. Mol. Biol. 73 (2000) 1-49.[2] L. Hecht, L.D. Barron, E.W. Blanch, A.F. Bell, L.A. Day, J .Raman Spectrosc. 30 (1999) 815-825.[3] P. Bour, T.A. Keiderling, J. Chem. Phys. 119 (2003), 11253-11262.
CONCLUSIONS
• Our goal was to find models suitable for simulation of Raman and ROA spectra of zwitterionic amino acids. To improve harmonic vibrational frequencies we have used a combination of the B3LYP and BPW91 functionals, COSMO continuous solvent model and systems with explicit water.
• ROA intensities are sensitive to majority of conformational changes. Some spectral features can be explained only by a presence of several conformers (band broadening etc.).
• The results suggest that the NH3
+ group is rotating freely, CH3 and COO– groups partially in Alanine and that Proline ring is very flexible.
ABSTRACT
Raman optical activity (ROA) measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right and left circularly polarized incident laser light. The ROA spectra of a wide range of biomolecules in aqueous solutions can be measured routinely. Because of its sensitivity to the chiral elements, ROA provides new information about solution structure and dynamics, complementary to that supplied by conventional spectroscopic techniques [1].
Incident circular polarization (ICP) ROA instrument has been built at the Institute of Physics following the design of the instrument constructed in Glasgow [2]. Combination of experimental and computational approaches represents unique and powerful tool for studying structure and interactions of biologically important molecules.
Computation of ROA is a complex process, including evaluation of equilibrium geometry, molecular force fields and polarizability tensor derivatives. In case of zwitterionic amino acids and peptides many complications arise also from their conformational flexibility and strong interaction with the solvent, which has to be taken into account in the modeling. For our ROA simulations we used continuum solvent models and solvation with explicit molecules of water [3].
Conformational space of L-alanine was investigated in detail by rotating the NH3+, CH3 and COO- groups. Our calculations suggest that NH3+ group is freely rotating while CH3 and COO- groups rotate only limitedly. Proline molecule contains a non-planar five-member ring and exhibits two major conformations with very similar energies. Conformational space of L-proline was examined by puckering the ring and also rotating COO- group. Weighted average spectra that were constructed can explain natural broadening of several spectral bands in particular in the low wavenumber region.
Finally we have shown that the simulation techniques requiring consideration of system dynamics and averaging over molecular conformations and solvent configurations are able to provide realistic ROA spectra of flexible and polar molecules.
EXPERIMENTAL
As an excitation source, a CW argon ion laser is employed. An improved linearly polarized radiation emerging from the polarizer passes through an electro-optic modulator (EOM), a longitudinal Pockels cell based on a potassium dideuteriumphosphate crystal. The EOM is driven by high-voltage linear differential amplifier. Right and left circular polarization states are generated by applying the appropriate voltages across the EOM electrodes. The circularly polarized laser beam is focused by plane-convex lens into a standard quartz cell containing typically 80-100 l of a sample. Before the sample is reached, the focused laser beam passes through holes drilled in a plane mirror, a collimating lens and a Lyot depolarizer. The backscattered radiation emerging from the sample is depolarized by the Lyot filter and then collimated by a lens. The collimated radiation is deflected by 90 with plane mirror and then focused by a camera lens onto an entrance slit of the single-stage stigmatic spectrograph ( f/1.4 ). A tilted holographic super notch filter is placed in front of the entrance slit to block the Rayleigh scattering. Spectrograph is equipped with a holographic transmission grating and the dispersed light is stored in a liquid nitrogen cooled back-illuminated CCD detection system based on EEV chip with high quantum efficiency having 1340 x 100 pixels.
Rotation of COO- group
0
1
2
3
4
5
6
7
0 30 60 90 120 150 180
Torsion Angle
Ener
gy [k
cal/m
ol]
konf B
konf A
Rotation of NH3+ group
0
0.5
1
1.5
2
2.5
3
3.5
-120 -100 -80 -60 -40 -20 0
Torsion Angle
Ene
rgy
[kca
l/mo
l]
Rotation of CH3 group
0
0.5
1
1.5
2
2.5
3
3.5
60 80 100 120 140 160 180
Torsion Angle
Ene
rgy
[kca
l/mo
l]
Rotation of COO- group
0
1
2
3
4
5
6
7
8
0 50 100 150 200
Torsion Angle
Ene
rgy
[kca
l/mo
l]
BPW91/6-31G**
BPW91/6-31++G**
BPW91/6-311G**
B3LYP/6-31G**
B3LYP/6-31++G**
B3LYP/6-311G**
ROA ICP experimental data. L-Alanine was dissolved in deionized water at final concentration of about 1.65 mol/L. Experimental parameters: laser wavelength 514.5 nm, laser power 440 mW, spectral resolution 6.5 cm-1, acquisition time 4 h.
Spectral Dependency - Rotation of NH3+ group :
L-Alanine
Conformational space of L-alanine was investigated in detail by rotating the NH3
+ (H-N-C-C*), CH3 ( H-C-C-C*) and COO- ( O-C*-C-N) groups. For each group one torsion angle was fixed and the rest was optimized.
L-Proline
Proline in H2O Proline in D2O
ROA ICP experimental data. L- and D-Proline was
dissolved in water at final concentration of about 3 M
and in D2O at 2 M. Experimental parameters:
laser wavelength 514.5 nm, laser power 440 mW, spectral resolution 6.5 cm-1, acquisition
time 6 h.
Experiment:
L-Pro and D-Pro
Rotation of COO- group.Average of all
conformers below Boltzmann Quantum,
below 1 kcal/mol and below 1.5 kcal/mol
(polar model)
Average of A (blue)
and B (red) Conformations.
Proline exhibits two major conformations very similar energies (E=0.3kcal/mol).
Conformational space of L-proline was investigated in detail by rotating COO- ( O-C*-C-N) group and puckering the ring – rotation around
AT9 ( C-C-C-N) torsion angle. Only one torsion angle was fixed and rest of the molecule was optimized.
Equilibrium geometries and harmonic force fields were calculated with the Gaussian program using the BPW91 DFT functional, base 6-31++G** and the COSMO solvent model. Optical activity tensors A and G’ was calculated in DALTON, HF/6-31++G** (in vacuum).
Equilibrium geometries and harmonic force fields were calculated with the Gaussian program using the BPW91 DFT functional, base 6-31++G** and the COSMO solvent model.
ROA tensors were calculated on HF/6-31++G** level in DALTON.
X Data
200 400 600 800 1000 1200 1400 1600 1800
Y D
ata
0
10
20
30
40
50
60
X Data
200 400 600 800 1000 1200 1400 1600 1800
Y D
ata
0
10
20
30
40
50
60
X Data
200 400 600 800 1000 1200 1400 1600 1800
Y D
ata
0
10
20
30
40
50
60
X Data
200 400 600 800 1000 1200 1400 1600 1800
Ram
an In
tens
ity
0
10
20
30
40
50
60
X Data
200 400 600 800 1000 1200 1400 1600 1800
Y D
ata
0
10
20
30
40
50
60
Wavenumber
300 600 900 1200 1500 1800
Ram
an
0
X Data
200 400 600 800 1000 1200 1400 1600 1800
Y D
ata
-200
-150
-100
-50
0
50
100
150
200
X Data
200 400 600 800 1000 1200 1400 1600 1800
Y D
ata
-200
-150
-100
-50
0
50
100
150
200
X Data
200 400 600 800 1000 1200 1400 1600 1800
Y D
ata
-200
-150
-100
-50
0
50
100
150
200
X Data
200 400 600 800 1000 1200 1400 1600 1800
RO
A In
tens
ity
-200
-150
-100
-50
0
50
100
150
200
X Data
200 400 600 800 1000 1200 1400 1600 1800
Y D
ata
-200
-150
-100
-50
0
50
100
150
200
Wvenumber
300 600 900 1200 1500 1800
Y D
ata
-200
-150
-100
-50
0
50
100
150
200
0°
-20°
-40°
-60°
-80°
-100°
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R +
I L
0
1e+9
2e+9
3e+9
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R -
I L
-4e+5
0
4e+5
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R +
I L
0
50
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
(IR -
I L)
. 104
-100
-50
0
50
100
Average Spectra from all conformation – free rotation of NH3
+ group is assumed.
Experiment
Calculation
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R +
I L
4e+9
8e+9
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R -
I L
-2e+6
0
2e+6
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R +
I L
0
20
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
(IR -
I L)
x 10
4
-100
0
100
2
34
56
7 8 910
11
12,1
3
14,15
16
17
18,19
202122
23
24 25
26,2
7,28
A28
B,2
930
,31
32,3
3
34
35 36
2
3 4B 6A
7A 8A
9A
1011
12,1
3
14,15
1617B
18,19
20A
20B
2122
2324
25
26-29
30,3
1B32
,33
34 35
36
2
3
4B
4A
5
6
7
8
9 10
11
12,1
3
1415
16
17
18
19
20
2122
23 24
25
26,2
7,28
A28
B,2
9
30,31
32,33
343536
2B
2A
3
4B
5
6A
7A8A
9A
1011B
11A
12,13
1415
16
17B
1819
20A
20B
21
22A
22B
23 24
25
26
27,28A
28B,29
30A
31A
32 34B
33A 34A 35
3630B,31B
Wavenumber
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R +
I L
0
1
2
3
Wavenumber (cm-1)
200 400 600 800 1000 1200 1400 1600 1800
(IR -
I L)
x 10
4
-15
0
15
Wavenumber
200 400 600 800 1000 1200 1400 1600
I R +
IL
4e+9
8e+9
Wavenumber
200 400 600 800 1000 1200 1400 1600
I R -
I L
-2e+6
0
2e+6
Wavenumber
200 400 600 800 1000 1200 1400 1600
I R +
I L
0
20
Wavenumber
200 400 600 800 1000 1200 1400 1600
(IR -
IL)
x 10
4
-100
0
100
2
3 4 56
7
8A
8B 9 10
11
12A
12B
,13B 13
A14
,15
16
17
18
1920
21
23,2
4
2627
28
29
30
31,3233,34
3536
25
22
2
3 4B
5
6
7 8A
8B 9
1011A
11B
12A
13 14,1
5
16
17,1
8
19B
20B
21
22A
23,2
4A
24B
2526
27
28
29
30
31B
31A
,32B
33
35
36
2B
2A
3
4
4B
4A
5B
5A
6
6
7
7
8A
8
8B9
9
10
11A
11B
12A
12B
13
14A
14B
,15
16
17,1
8
11A
12,1
3
14,1
5
16B
,17
18
19
20,2
122
A
22A
21
19B
19A
20B
23 24,2
5
26,2
7
28
29
31,3
2
33,34
3536
22B
23
24A
24B
25B
26B
,27B
26A
,27A 28
,29
30 31B
,32A
31A,32B
33
35
34A
36
2
5
10
3
Wavenumber
200 400 600 800 1000 1200 1400 1600
I R +
I L
0
2
4
Wavenumber (cm-1)
200 400 600 800 1000 1200 1400 1600
(IR -
I L)
x 10
4
-10
0
10
AT9
-60 -40 -20 0 20 40 60
H (
kca
l/mo
l)
-2
0
2
4
6
8
10
12
AT9
-60 -40 -20 0 20 40 60
H +
V(f
)/2
(k
cal/m
ol)
0
1
2
3
4
5
6
Energy dependencies (different DFT functionals and basis sets):
Average spectra :
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R +
I L
0
20
40
Wavenumber (cm-1)
200 400 600 800 1000 1200 1400 1600 1800
(IR -
I L)
x 104
-150
0
150
Wavenumber
200 400 600 800 1000 1200 1400 1600 1800
I R +
I L
0
20
Wavenumber (cm-1)
200 400 600 800 1000 1200 1400 1600 1800
(IR -
I L)
x 104
-100
0
100
Rotation of AT9 torsion angle - ring puckering:
Average of all conformers
(Maxwell-Boltzmann statistics):
F(a)=A Exp(-Ea/k.T)
averageCOO- group
A+B
Averagering puckering
AverageExplicit water
Average of 4 conformations
calculated in vacuum with explicit water
molecules
Example of cavity around proline constructed by COSMO model. Color corresponds to charge induced by molecule to
the surface
averageCOO- group
A+B