Monitoring of Zwitterionic Proline and Alanine Conformational Space by Raman Optical Activity

1. Monitoring of Zwitterionic Proline and Alanine Conformational Space by Raman Optical Activity Josef Kapitán a,b, Petr Bouř band Vladimír Baumruk a aInstitute of Physics, Charles University, Ke Karlovu 5, Prague, 12116, Czech Republic bInstitute 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 (DE=0.3kcal/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 6-31++G** and the COSMO solvent model. ROA tensors were calculated on HF/6-31++G** 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 6-31++G** and the COSMO solvent model. Optical activity tensors A and G’ was calculated in DALTON, HF/6-31++G** (in vacuum). A+B A+B 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 Average Spectra from all conformation – free rotation of NH3+ group is assumed. -100° 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.