Circular Dichroism in Protein Analysis
71
of membrane proteins (determined by
X-ray diffraction), of which there are rel-
atively few. Nevertheless, it is possible
to estimate secondary structures content
from CD spectra of membrane proteins
using approaches such as convex con-
straint analysis. Five component spectra
have been identi±ed. They are two differ-
ent types of
α
-helices (the
α
-helix in the
soluble domain and the
α
T
-helix for trans-
membrane
α
-helix),
β
-sheet,
β
-turn, and
unordered conformation. The character-
istics of CD spectra of
α
-helix,
β
-sheet,
β
-turn, and random coil have been dis-
cussed above. The
α
T
-helix has a positive
band in the range of 195 to 200 nm.
The intensity of the 208-nm band is
slightly more negative than that of the
222-nm band.
α
T
-helix has a larger rota-
tional strength than an
α
-helix in aqueous
conditions. Membrane proteins are im-
mersed in a much lower dielectric medium
than soluble proteins, which may explain
some differences. Another important fac-
tor may be that the average chain length
of an
α
T
-helix is 25 residues, which is
about twice that of an
α
-helix in a solu-
ble domain.
CD spectra can give much informa-
tion about membrane proteins, such as
secondary structure, fold motifs, confor-
mational changes, environmental effects,
folding, and insertion into membranes.
However, there are potential artifacts of
differential scattering, absorption flatten-
ing, and wavelength shifts, which may
affect the CD spectra. CD spectra of
membrane proteins often exhibit various
degrees of distortions in shapes, intensi-
ties, and/or positions of the CD bands, and
shifts in crossover points. There have been
several experimental and theoretical ap-
proaches for differentiating the CD bands
from the artifacts of the membrane protein
CD spectra.
7
Conclusion and Outlook
CD spectroscopy is a useful technique
for detecting protein secondary structure
quickly and quantitatively. It is also useful
in
following
protein
folding/unfolding
processes with stopped-flow or faster time-
resolved techniques. The development of
transient VCD may present a signi±cant
advance in quantifying the timescales of
motions of biological systems. Currently,
there is ongoing research to improve the
accuracy of estimates of protein secondary
structure content. Making such estimates
more quantitative remains a challenge,
as
there
are
many
factors
that
may
affect the intensity and wavelength of
CD spectra. Temperature and solvents
are important external factors; dihedral
angles and hydrogen-bonding patterns are
key internal factors. All of these can
change the relative orientation of n
π
and
ππ
transitions, which in turn can lead
to different CD spectra. In general, CD
spectroscopy is a rapid technique, which
is sensitive to protein secondary structure.
More reliable analysis of CD spectra may
emerge from ongoing experimental and
theoretical studies.
See also
Chirality in Biology.
Bibliography
Books and Reviews
Berova, N., Nakanishi, K., Woody, R.W. (1994)
Circular Dichroism: Principles and Applications
,
VCH Publishers Inc., New York.
Fasman, G.D. (1996)
Circular Dichroism and
the Conformational Analysis of Biomolecules
,
Plenum Press, New York.
Green±eld, N.J. (1996) Methods to estimate the
conformation of proteins and polypeptides
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