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