1. Molecular Spectroscopy 2008 VIBRATIONAL SPECTROSCOPY OF TRANSFER RNA OF E.COLI: EXPERIMENT and MODELING Tatiana Globus, Alexei Bykhovski, Tatyana Khromova, Boris Gelmont Department of Electrical and Computer Engineering, University of Virginia,Charlottesville, VA 22908. Sponsored by the ARMY, DTRA Joint Science and Technology Office for Chemical Biological Defense Detection Capability Area, DTRA/CBT, under Contract DAAD19- 00-1-0402, in part by U.S.Army NGIC Contract # DASC01-01-C0009, and by a grant from the W. M. Keck Foundation

2. Molecular Spectroscopy 2008 Outline I. Progress in Low-THz FTIR vibrational spectroscopy of biological macromolecules. Examle: experimental spectra (dilute solutions of transfer RNA (tRNA) molecule of E. coli II. Progress in computational modeling of biological macromolecules. Examle: modeling results (transfer RNA (tRNA) molecule of E. coli) Photonics West 2006, Biomedical Optics, San Jose, California, 21-26 January 2006 SPIE, Orlando 2006

3. Molecular Spectroscopy 2008 FTIR Fourier Transform Spectroscopy  Bruker IFS-66 spectrometer (Hg-lamp source, He cooled Si-bolometer @ 1.7 ºK for signal detection, three vacuum systems are not shown).  Range of interest throughout 10 cm -1 – 25 cm -1 Fourier transform (FT) transmission spectroscopy provides till now the most detailed information on THz vibrational spectral signatures of bio-simulants. THz transmission (absorption) spectra taken with a good resolution of 0.25 cm -1 reveal multiple resonances in the sub-THz range.

4. Molecular Spectroscopy 2008 Resonance Fourier-Transform THz spectroscopy A commercial FTS (Bruker IFS-66) system with  a Si-bolometer operating at T = 1.7 o K for the signal detection  a Hg- lamp as a source ( < 100 cm -1 )  Spectral resolution at least 0.25 cm -1  High sensitivity and reproducibility: standard deviation for transmission better than 0.3% (peak at 18.6 cm -1 is due to water vapor absorption) What is important? Good Instrument performance:  High transparency of substrates and windows  Sample chamber is not evacuated  Standard sample holder Beamsplitter125 μm Mylar Acquisition modeSingle sided, fast return Scan velocity80 kHz Resolution0.25 cm -1 Phase resolution2 cm -1 Zerofilling factor4 Phase correction modeMertz Apodization functionBlackman-Harris 3 terms High frequency limit<8000 cm -1 Spectrometer and Software Settings Spectral features at the level of 0.5-2% for transmission could be well resolved and reproducibly recorded.

5. Molecular Spectroscopy 2008 Challenges in experimental low THz vibrational spectroscopy  There are different sources of spectral artifacts.  The measurement system layout can be one of the sources due to multiple reflection between system elements.  Artifacts are closely related to the spectral resolution: the better the resolution – the stronger artifacts.  For vibrational spectroscopic characterization of materials, the required spectral resolution is dictated by the spectral width of vibrational modes that can be used for fingerprinting and have to be detected.  Sample preparation technique, including sample geometry (thickness), sample cell or sample substrate, can be the other source of spectral artifacts. The physical origin of these artifacts can be again caused by multiple reflection at interfaces. Proper choice of a sample preparation procedure and a sample cell geometry can eliminate or significantly reduce these effects.  Water and gases absorption lines present in the spectral range of interest can make reliable measurements extremely difficult because of mismatch between the reference (background) and sample measurements.

6. Molecular Spectroscopy 2008 Sample preparation Sample cell is assembled of two substrate (or window) thin films and a PE spacer between.  Substrates: polyvinylidene chloride (PVDC) films (Saran Film®Saran, the Dow Substrate thickness: 12 and 6 µm thick respectively to avoid interference (etalon) effects in these cell components.  The thickness of the spacer was 12  m, and the inner diameter was 18 mm.  Due to the surface tension, substrate films keep thin layers of solution between them even without sealing.  Less than 10  l of solution per sample. Samples were prepared in the form of water solutions (suspension) with the concentrations in the range 0.01-1 mg/ml.

7. Molecular Spectroscopy 2008 Liquid water and cell windows (substrates) absorb and contribute to the background.  Water absorption increases and the transmission decreases with increasing frequency.  The different spectra correspond to different thickness of water layer between two substrates.  DNA spectra were calculated against the transmission of cells with matching amounts of pure water between the corresponding substrates by dividing the measured spectrum of an assembled cell by the spectrum of a water cell with the same or next higher level of transmission.  The resulting absolute level of transmission is accurate within the limit of several %), because the amount of water in the reference spectrum does not exactly match the amount of water in the measured sample.  We are interested in resonance features of spectra - frequencies of vibrational modes. Water and substrate materials contributions

8. Molecular Spectroscopy 2008 Transfer RNA for tyrosine of E. coli Transfer RNAs (tRNA) are low molecular weight form of RNA which serves as an amino acid acceptor. The molecular structure of tRNA has a specific 3- dimensional conformation ( cloverleaf ). It has been demonstrated that the native cloverleaf conformation is essential for their biological activity.

9. Molecular Spectroscopy 2008 Model Description The initial atomic structure of tRNA for tyrosine was taken from X-ray data collected for crystallized molecules [T.KOBAYASHI, et al., NAT.STRUCT.BIOL., V. 10 425 2003]. The molecular structure was optimized using the potential energy minimization and molecular dynamical (MD) simulations (Amber). The effect of a liquid content of a bacterial cell was taken into account explicitly via the simulation of water molecules using TIP3P water model. This water model represents a rigid water monomer with three interaction sites. Coulomb and Lennard-Jones potentials are taken into account in TIP3P model. Structure Optimization

10. Molecular Spectroscopy 2008 MD simulations Biological molecule (blue) surrounded by water molecules (red).

11. Molecular Spectroscopy 2008 We have prepared samples and measured spectra of tyrosine tRNA in the form of very diluted solutions sealed between substrates made from polyethylene (Saran film, 12 mm thick). The results are obtained after dividing measured spectra by measured transmission of pure water contained between the same substrate films. EXPERIMENTAL RESULTS Spectra of tRNA for tyrosine solutions with 3 different concentrations between two Saran films. Averaged result is also shown.

12. Molecular Spectroscopy 2008 EXPERIMENT V THEORY The average absorption coefficient: the experiment for saran film (dotted green) and the theory, explicit water model, (solid blue). Theoretical estimate of the absolute value of the absorption coefficient in a diluted water solution ~ 1 cm -1 /1g/l

13. Molecular Spectroscopy 2008 CONCLUSIONS  We have demonstrated that Fourier Transform spectroscopy can be used to measure low frequency vibrational modes of biomolecules in dilute water solutions at the THz frequencies 10-25 cm -1.  Samples were prepared in the form of thin layers (~10  m) of water solutions (suspension) with the concentrations in the range 0.01-1 mg/ml.  The molecular structure of the entire transfer RNA (tRNA) molecule of E. coli was simulated and the associated THz vibrational spectra was derived theoretically. The molecular structure was optimized using the potential energy minimization and molecular dynamical (MD) simulations. Solvation effects (water molecules) were also included explicitly in the MD simulations.  The calculated THz signature of the tRNA of E. coli reproduces many features of measured spectra.