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How it works
Circularly polarized light spins about its direction of
propagation—right-handed light spins clockwise, while
le -handed light spins counter-clockwise. In a CD
experiment, a protein is irradiated with alternating pulses
of le and right circularly polarized light. If the protein is
chiral (non-symmetrical), it will absorb le and right light
to different extents. Consequently, a protein's secondary
structures have distinct spectral signatures. Therefore,
CD can be used to measure the general composition of a
protein, e.g., the fraction of the molecule that is composed
of different secondary structures, such as alpha helices
and beta-sheets. A scientist will first measure the spectral
signature of a protein in its native state, and then measure
it again a er a change in temperature or following
exposure to a denaturing agent to determine its effects on
the protein's structure.
Strengths
CD can measure multiple samples containing 20µg or less
of protein in aqueous solutions containing physiological
buffers in just a few hours. Small sample volumes are used
in CD.
Weaknesses
CD cannot determine where secondary structures are
located within a protein or how many there are. It can
only assess the general proportion of the protein that is
composed of these structures. Also, common aqueous
buffers o en absorb light in the range where proteins
exhibit differences in their absorption of circularly polarized
light. This technique is typically incompatible with
phosphate, carbonate, acetate, and sulfate buffers unless
they are extremely diluted.
Conclusion
Although CD does not have the same resolution as
crystallography, and therefore cannot define protein
structures, it provides a quick, flexible method for
measuring changes in protein folding following exposure
to both thermal and chemical stress.
Circular dichroism (CD) is a widely used method for assaying protein stability. It uses circularly polarized light to measure
both chemical or thermal denaturation.
Circular Dichroism
