K. Chruszcz-Lipska and E. W. Blanch
64
shot’ spectra from all of the distinct conformations present at equilibrium. however,
as the RoA observables depend on absolute chirality, the spectral contributions
from enantiomeric structures adopted by a mobile biopolymer exploring the full
range of accessible conformations available tend to cancel each other out. therefore, RoA exhibits a strong sensitivity to the dynamics of biomolecular structure
and more conformationally mobile structures display considerably reduced RoA
intensities compared to rigid biomolecules. this sensitivity contrasts with that of
conventional Raman band intensities which are ‘blind’ to chirality and so are generally additive and consequently less sensitive to conformational mobility. Although
ultraviolet circular dichroism (uvCd) also demonstrates an enhanced sensitivity to
the dynamics of chiral structures, the level of sensitivity of uvCd is lower than that
for RoA due to the former’s dependence on electronic transitions.
4.2 Instrumentation and Measurement
As discussed above, a backscattering geometry is the most favourable one for routine measurement of RoA spectra of biomolecules in aqueous solution. Backscattering RoA measurements can be made using either the ICP, SCP or other strategies
mentioned above, with the instrumentation required being different in each case.
up until around 2004–2005, most of the reported RoA spectra of biomolecules
were measured on ICP backscattering instruments at the university of glasgow in
the Barron laboratory. A detailed description of the optical layout of typical glasgow
backscattering ICP RoA instruments can be found elsewhere [24]. In summary, a
visible argon ion laser beam at 514.5 nm or a frequency-doubled Nd:YAg laser
beam at 532 nm is weakly focused into the sample solution contained in a small
rectangular fused quartz cell. the cone of backscattered light is reflected off a 45°
mirror, which has a small central hole drilled to allow passage of the incident laser
beam, through an edge filter to remove the Rayleigh line and into the collection
optics of a single grating spectrograph, a customized Kaiser holospec fast imaging
spectrograph. the detector is a Peltier-effect cooled back-thinned charge coupled
device (CCd) camera with a quantum efficiency of ~ 80 % over the spectral range
used and operating in multichannel mode to allow the full spectral range to be measured in a single acquisition. Spectral acquisition is synchronized with the electrooptic modulator used to switch the state of polarization of the incident laser beam
between right and left circular at a suitable rate. Counter-rotating half-wave plates
inserted in the incident laser beam after the electro-optic modulater average the azimuths of any residual linear contaminants in the right- and left-circular polarization
states of the beam and suppress artefacts arising from linear birefringence. Spectra
are displayed in analog-to-digital converter units as a function of the Stokes Raman
wavenumber shift with respect to the exciting laser line. typical laser power at
the sample is ~ 700 mW and sample concentrations of proteins, carbohydrates and
nucleic acids are ~ 30–100 mg/ml while those of intact viruses are ~ 5–30 mg/ml.
under these conditions RoA spectra over the range ~ 600–1700 cm
−1
are typically
obtained in ~ 5–24 hours for proteins and nucleic acids and ~ 1–4 days for intact
64
shot’ spectra from all of the distinct conformations present at equilibrium. however,
as the RoA observables depend on absolute chirality, the spectral contributions
from enantiomeric structures adopted by a mobile biopolymer exploring the full
range of accessible conformations available tend to cancel each other out. therefore, RoA exhibits a strong sensitivity to the dynamics of biomolecular structure
and more conformationally mobile structures display considerably reduced RoA
intensities compared to rigid biomolecules. this sensitivity contrasts with that of
conventional Raman band intensities which are ‘blind’ to chirality and so are generally additive and consequently less sensitive to conformational mobility. Although
ultraviolet circular dichroism (uvCd) also demonstrates an enhanced sensitivity to
the dynamics of chiral structures, the level of sensitivity of uvCd is lower than that
for RoA due to the former’s dependence on electronic transitions.
4.2 Instrumentation and Measurement
As discussed above, a backscattering geometry is the most favourable one for routine measurement of RoA spectra of biomolecules in aqueous solution. Backscattering RoA measurements can be made using either the ICP, SCP or other strategies
mentioned above, with the instrumentation required being different in each case.
up until around 2004–2005, most of the reported RoA spectra of biomolecules
were measured on ICP backscattering instruments at the university of glasgow in
the Barron laboratory. A detailed description of the optical layout of typical glasgow
backscattering ICP RoA instruments can be found elsewhere [24]. In summary, a
visible argon ion laser beam at 514.5 nm or a frequency-doubled Nd:YAg laser
beam at 532 nm is weakly focused into the sample solution contained in a small
rectangular fused quartz cell. the cone of backscattered light is reflected off a 45°
mirror, which has a small central hole drilled to allow passage of the incident laser
beam, through an edge filter to remove the Rayleigh line and into the collection
optics of a single grating spectrograph, a customized Kaiser holospec fast imaging
spectrograph. the detector is a Peltier-effect cooled back-thinned charge coupled
device (CCd) camera with a quantum efficiency of ~ 80 % over the spectral range
used and operating in multichannel mode to allow the full spectral range to be measured in a single acquisition. Spectral acquisition is synchronized with the electrooptic modulator used to switch the state of polarization of the incident laser beam
between right and left circular at a suitable rate. Counter-rotating half-wave plates
inserted in the incident laser beam after the electro-optic modulater average the azimuths of any residual linear contaminants in the right- and left-circular polarization
states of the beam and suppress artefacts arising from linear birefringence. Spectra
are displayed in analog-to-digital converter units as a function of the Stokes Raman
wavenumber shift with respect to the exciting laser line. typical laser power at
the sample is ~ 700 mW and sample concentrations of proteins, carbohydrates and
nucleic acids are ~ 30–100 mg/ml while those of intact viruses are ~ 5–30 mg/ml.
under these conditions RoA spectra over the range ~ 600–1700 cm
−1
are typically
obtained in ~ 5–24 hours for proteins and nucleic acids and ~ 1–4 days for intact