Raman optical activity instrument for studies of biopolymer structure and dynamics

Abstract

The latest version of a multi-channel Raman optical activity (ROA) instrument implementing incident circular polarization (ICP) modulation within a backscattering geometry and optimized for measurements on biopolymers in aqueous solution is described. It is based on a fast single-stage, imaging (stigmatic) monochromator equipped with a high optical density holographic notch filter, a holographic transmission grating and a thinned back-illuminated thermoelectrically cooled charge-coupled device (CCD) detector with a high quantum efficiency. A large-aperture longitudinal electro-optic modulator (Pockels cell) is employed to switch between orthogonal circular polarization states in the incident laser radiation. A thick Lyot depolarizer is used for depolarization of the backscattered Raman radiation. This backscattering ICP CCD ROA design is currently realized with two prototype instruments dedicated to studies of the structure and dynamics of biopolymers in aqueous solution. Backscattered ICP ROA spectra of the following samples are presented as typical examples of the excellent performance characteristics: poly(L-glutamic acid) in ahelical and disordered conformations; bovine a-lactalbumin in native and acid molten globule states; human immunoglobulin; calf thymus DNA and both magnesium-bound and magnesium-free phenylalanine-specific transfer RNA; and filamentous bacterial viruses Pf1 and M13.

Raman Optical Activity Demonstrates Poly(l-proline) II Helix in the N-terminal Region of the Ovine Prion Protein: Implications for Function and Misfunction

“…65 ROA is measured by synchronising the Raman spectral acquisition with an electro-optic modulator to switch the polarisation state of the incident argon-ion laser beam between right and left circular at a suitable rate. The spectra are displayed in analog-to-digital counter units as a function of the Stokes wave number shift with respect to the exciting laser wave number.…”

Raman optical activity characterization of native and molten globule states of equine lysozyme: Comparison with hen lysozyme and bovine α-lactalbumin

“…33,34 ROA is measured by synchronizing the Raman spectral acquisition with an electrooptic modulator that switches the polarization of the incident argon ion laser beam between right and left circular at a suitable rate. The spectra are displayed in analog to digital counter units as a function of the Stokes Raman wave number shift with respect to the exciting laser wavelength.…”

Solution structure of native proteins with irregular folds from Raman optical activity

“…ROA has recently been applied to biomolecules and provides a new perspective on their solution structure and dynamics. [5][6][7][8] ROA bears the same relation to conventional Raman spectroscopy as does uv CD to conventional uv absorption spectroscopy. ROA is able to cut through the complexity of the conventional vibrational spectra of biopolymers since the largest ROA signals are often associated with vibrational coordinates that sample the most rigid and chiral parts of the structure.…”