Introduction to VCD in
Biopolymer studies
Electronic
circular dichroism in the ultraviolet (ECD) has become an indispensable
tool for qualitative characterization of proteins in solution.
However accessible amide transitions are limited in number, are broad and
overlapping, and the resulting UV spectral bands are often sensitive to
environmental or local perturbations. Vibrational spectroscopies, such as
infrared (IR) and Raman, also have an established role for
characterization of secondary structures of proteins and peptides. While
exhibiting many resolved transitions, these are generally limited to
measurement of relatively small frequency shifts characteristic of the
effects of conformation on bond strengths or to perturbations due to
hydrogen bonding. Vibrational CD (VCD) [and Raman optical activity, not
developed yet for proteins in our lab, see Laurence Barron, Glasgow Univ.]
has developed as a hybrid of these techniques and found application in
the biomolecular structural studies (1). VCD can be used to correlate
data for several different spectrally resolved transitions that involve
different localized vibrations of the molecule; and each of these features
will have a distinct band shape dependence on molecular
stereochemistry. Empirical correlation of spectral features with
secondary structure has historically been the most profitable route for
stereochemical utilization of both electronic CD and vibrational(IR
and
Raman) spectroscopies. The difference in the origins of CD measured in the two spectral regions suggests that they would bear a complementary relationship that could enhance the quality and quantity of structural information derivable from eit
her one alone and compensate for shortcomings of each. A series of studies on peptides of varying sequence and length have borne this out. VCD has a distinctively shorter length dependence that does ECD which leads to its having more sensitivity to the
variety of secondary structure types seen in proteins (1). Quantitative
approaches to a uniform systematic analysis of VCD, FTIR and ECD data,
with the eventual goal of carrying out a coupled analysis, is a major
topic of our ongoing studies. Experimentally, VCD spectra are
routinely measured on either dispersive or FTIR-based instruments, both of
which have been described in detail in the literature (2). To date,
protein dispersive VCD obtained at ~10 cm-1 resolution by averaging
several repetitive scans over the band of interest have a
signal-to-noise ratio (S/N) advantage over FTIR-VCD spectra (3). Our spectra are usually obtained on very concentrated (up to50 mg/ml in D2O and 200 mg/ml in H2O) solutions in short path length (25 µ m and down
to 6 µm for H2O) sample cells with CaF2 windows (4). In non-aqueous
environments, for model peptides or solvent perturbation tests of protein
structure, lower concentrations and longer path lengths are possible. For
purposes of comparison and further spectral analyses, higher
resolution and better S/N FTIR absorption spectra are obtained on the same or more dilute samples, and ECD spectra (>180 nm) are obtained with much more dilute samples. All spectra are systematically treated using the SpectraCalc
package of programs for data manipulation.
--Research Topics--MVCD--Biopolymers--Instrumentation--Small
Molecules--Other Interests--Keiderling Home--
