Raman Optical Activity (ROA)

ROA has been a minor, but long term interest of the Keiderling research group. Due to its complementarity to VCD, ROA has a place in our theoretical and experimental efforts. However, recently, the necessary technology to support our research interest in biopoloymers has gotten ahead of our funding and so that ROA is not used much for our present research. Normal Raman spectra still do have a role and the ROA spectrometer we have constructed has been easily modified to provide a good vehicle for getting Raman spectra for proteins and other systems. ROA has been (and remains) the focus of Laurence Barron's lab (Glasgow) following his initial development of reliable ROA when a postdoc with David Buckingham at Cambridge. Werner Hug at Fribourg and the Nafie, Diem, Polavarapu and Keiderling labs have also been active in this area, at various times. Most recently, a commercial ROA spectrometer has been announced by BioTools Inc. based on a novel design of Werner Hug. This instrument has exceptional baseline qualities and a very good S/N, potentially making it ideal for biopolymer studies.

ROA, the differential scattering of left and right circularly polarized light shifted by molecular vibrations, or DI=IR-IL, is both a weaker and stronger than VCD, since it is based on Raman scattering, which is reduced by ~10-6 from the input laser intensity, ROA bust be viewed as weak. However, one detects Raman scatter (like fluorescence) against a null background, so very high sensitivity detectors can be sued, and the exciting and scattered light are in the visible, simplifying and generalizing optics and detection schemes. . Reviews of ROA abound (36-41). A key impetus for ROA in the vibrational region of the spectrum is that it is rich with resolved transitions, which are mostly all detectable. ROA normally can access the entire "mid-IR" part of the vibrational spectrum, from 2000 to a few hundred cm-1, and water in particular is a poor scatterer, so studies of biomolecules in their natural solvent are quite possible. The richness of ROA spectra coupled with the transitons being characteristic of localized parts of the molecule, and their sign patterns being related to the local stereochemistry, makes ROA a technique worth considering, despite its technological difficulties .

Experimentally, the best quality and broadest range of ROA for biomolecules is collected in the Barron lab in a 180o backscattering experiment. The output of a Ar ion laser (typically 514.5 nm) is polarized and modulated between left and right circular with a pockels cell. This modulation is slow as the counts in each polarization state are accumulated and stored in separate channels for eventually subtraction to get DI. Thus unlike most VCD experiments, dependent on analog demodulation of the signal, ROA is typically a fully digital experiment. In 180o backscattering, the laser is focussed onto the surface of the sample, typically in a quartz cuvette, and the scattered light is collected by a fast lens after passing through a Lyot depolarizer, onto a mirror at 45i to reflect the light into the detections optics. The optics that overlap input and output beams are drilled with a small hole to pass the laser and collect the scatter (big cone). In Barron's lab the detection uses a fast monochromator, an f/1.8 Kaiser transmission grating device, and a back-thinned CCD which detects the signal and digitizes it for the computer control. His lab is able to collect ordinary ROA of neat liquids in minutes and gets ROA of biopolymers in times of the order of an hour, sometimes less, sometimes more depending on concentration and the interference scatter of solvent and impurities. As compared to VCD, ROA is less flexible in terms of concentration, lengthening the path is not an option, so to make dilution feasible, higher sensitivity is needed, a feat partially achieved by the Barron and Hug labs.. For comparison, VCD, being absorptive, can dilute the sample and lengthen the path. With H2O and proteins this is not very practical , but with D2O and proteins or organic solvents and most any sample, it works well.

To provide a reference to the Keiderling lab ROA, In summary, we use a Coherent Innova 300 Ar laser, with up to 1 W in 488 and 514 nm lines, a KDP Pockels cell controlled by a variable HV supply under computer control. Our 180o backscatter sampling setup is much as described above and the scattered Raman light is passed through a holographic notch filter (to eliminate the unshifted laser scatter) and passed to a 0.64 m J-Y monochromator with a PAR OMA III photodiode array. This is intensified and cooled for high sensitivity. The detection system and speed of the monochromator in this older design, do limit sensitivity. We can obtain high quality ROA of neat liquids in the order of 1 hour, but measuring ROA of biopolymers in water becomes extremely time consuming resulting in marginal S/N. This instrument was origninally developed by Mark Vavra who studied a series of chiral ahcohols (1) and upgraded by Vladimir Baumruk and Cheok Tam who published a few results on small molecules using it.(2,3,4)

An important characteristic of ROA was demonstrated by Tam (2), in that teh chirality sensitivity fo RAO is shorter range than even VCD. It seems, and this holds also for protein data from the Barron lab, that ROA senses the local stereochemistry, in essence the electronic structure and its coupling to nuclear motion leading to the polarizability around the chiral center. In proteins this is evident in the main transitions, amide I and III, tending to have the same sign in all conformations but shifting and changing intensity with structural variation. This sensitivity comes form the basic f,y variation of the electronic structure at the sensitive vibrational site. If interpretable, this is a direct conduit to secondary structure information. The resolution of these transitions and the sensitivity to many more transitions gives ROA its impact in protein conformational studies.

 

1. Mark Vavra thesis and "Raman Optical Activity of Small Molecules on a Spectrometer Assembled from Commercially Available Components,"M. R. Vavra, T. A. Keiderling, in "Eleventh International Conference on Raman Spectroscopy," ed. R. J. H. Clark and D. A. Long, John Wiley, Chichester, U.K., p. 973-974, (1988).

2. "An Experimental Comparison of Vibrational Circular Dichroism and Raman Optical Activity Using 1-amino-2-propanol and 2-amino-1-propanol as Model Compounds" Cheok N. Tam, Petr Bour and Timothy A. Keiderling, Journal of the American Chemical Society 1 19 7061-7064 (1997).

3. "Vibrational optical activity of (3S,6S)-3,6-dimethyl-1,4-dioxane-2,5-dione" C. N. Tam, P. Bour, T. A. Keiderling, Journal of the American Chemical Society 118, 10285-10293 (1996).

4. "Vibrational optical activity Study of trans-Succinic-d2 anhydride." Petr Bour, Cheok Tam, M. Shaharuzzaman, J. S. Chickos, T. A. Keiderling, Journal of Physical Chemistry 100, 15041-15048 (1996)

36. LD Barron. Vibrational Spectra and Structure 17B: 343-368, 1989.

37. PL Polavarapu. Vibrational Spectra and Structure 17B: 319-342, 1989.

38. LD Barron, L Hecht. In: Clark, R. J. H., Hester, R. E., eds. Biomolecular Spectroscopy, Part B. : John Wiley and Sons, 1993, pp 235-266.

39. LA Nafie. In: Evans, M., Kielich, S., eds. Modern Nonlinear Optics, Part 3. New York: Wiley, 1994, pp 105-206.

40. LD Barron, L Hecht, AD Bell. In: Fasman, G. D., ed. Circular Dichroism and the Conformational Analysis of Biomolecules. New York: Plenum, 1996, pp 653-695.

41. LA Nafie. Annu Rev Phys Chem 48: 357-386, 1997.