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Monitoring of Zwitterionic Proline and Alanine
Conformational Space by Raman Optical
Activity Josef Kapitán a,b, Petr Bour b and
Vladimír Baumruk a a Institute of Physics,
Charles University, Ke Karlovu 5, Prague, 12116,
Czech Republic b Institute of Organic Chemistry
and Biochemistry, Flemingovo nám. 2, Prague,
16610, Czech Republic
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 ml 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.
L-Proline
L-Alanine
Proline exhibits two major conformations very
similar energies (DE0.3kcal/mol).
Conformational space of L-proline was
investigated in detail by rotating COO- (?
O-C-Ca-N) group and puckering the ring
rotation around AT9 (? Cg-Cb-Ca-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 6-31G and the
COSMO solvent model. ROA tensors were
calculated on HF/6-31G level in DALTON.
Proline in H2O
Proline in D2O
Conformational space of L-alanine was
investigated in detail by rotating the NH3 (?
H-N-Ca-C), CH3 (? H-C-Ca-C) and COO- (?
O-C-Ca-N) groups. For each group one torsion
angle was fixed and the rest was optimized.
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.
Energy dependencies (different DFT functionals
and basis sets)
Experiment L-Pro and D-Pro
Equilibrium geometries and harmonic force fields
were calculated with the Gaussian program using
the BPW91 DFT functional, base 6-31G and the
COSMO solvent model. Optical activity tensors A
and G was calculated in DALTON, HF/6-31G (in
vacuum).
AB
AB
Average of A (blue) and B (red) Conformations.
Spectral Dependency - Rotation of NH3 group
average COO- group
average COO- group
0
Rotation of COO- group. Average of all
conformers below Boltzmann Quantum, below 1
kcal/mol and below 1.5 kcal/mol (polar model)
Average spectra
Experiment
-20
-40
Rotation of AT9 torsion angle - ring puckering
Average ring puckering
Example of cavity around proline constructed by
COSMO model. Color corresponds to charge induced
by molecule to the surface
Calculation
-60
Average of all conformers (Maxwell-Boltzmann
statistics) F(a)A Exp(-Ea/k.T)
-80
Average Explicit water
-100
Average Spectra from all conformation free
rotation of NH3 group is assumed.
Average of 4 conformations calculated in vacuum
with explicit water molecules
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.

• 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.

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.