70
Circular Dichroism in Protein Analysis
number of different conformers, with the
relative geometry of nearest neighbors de-
termined statistically. Another opinion is
that fully ionized poly(Glu) and poly(Lys) at
low temperature and low ionic strengths
are in an extended helix conformation,
which is similar to the left-handed 3
1
helix
of poly(Pro)II helix. Theoretical calcula-
tions suggest that the parallel-polarized
ππ
transition occurs as a positive peak
at 209 nm and that the perpendicular-
polarized component is at 201 nm with
a negative rotational strength. In general,
random coil structures with different se-
quences and under different conditions
can give a variety of CD curves. However,
it seems likely that unordered polypeptides
that have a positive band near 218 nm may
have a substantial amount of poly(Pro)II
helical conformation.
6.6
Polyproline Helices
The homopolymer poly(Pro) can adopt
two different conformations. Poly(Pro) I
is a right-handed helix with
cis
-peptide
bonds throughout and it is stable only
in relatively nonpolar solvents such as
n
-propanol. Poly(Pro)II is a left-handed 3
1
-
fold helix with trans residues. These two
types of polyproline helices have different
CD spectra. Poly(Pro)I has a strong positive
band at
215 nm and a slightly weaker
negative at
195 nm. Poly(Pro)II has a
weak positive band at
230 nm and a
strong
negative
band
at
205 nm
of
ellipticity
∼−
60 000 deg cm
2
dmol
1
.
6.7
Extrinsic Chromophores
The CD spectra above 300 nm can be
used to study transitions in chromophoric
prosthetic groups, metal ions, inhibitors,
or substrate analogues. Proteins lacking
prosthetic groups do not exhibit absorp-
tion or CD bands above 300 nm, except
possibly for the tail of a disulFde tran-
sition or a tryptophan band just above
300 nm. The CD spectra of disulFdes
have a characteristic tail stretching be-
yond 300 nm. There are three mechanisms
contributing to the CD of chromophores
bound to proteins. An extrinsic chro-
mophore may be inherently chiral. There
may be coupling between electronic tran-
sitions on the extrinsic chromophore and
chromophores in the protein. It is also
possible that transitions of differing sym-
metry on the extrinsic chromophore may
mix
because
of
the
electrostatic
Feld
of
the
protein.
The
last
two
mecha-
nisms depend on both the extrinsic chro-
mophore geometry and also the structure
of the protein.
6.8
Membrane Proteins
There are two kinds of membrane pro-
teins: extrinsic (or peripheral) and intrinsic
(or integral). Extrinsic membrane proteins
may be removed from the membrane, or
solubilized, by mild treatment, such as
shaking with a dilute salt solution. Intrin-
sic membrane proteins cannot be removed
from the membrane without treatment
that destroys the membrane structure,
such as dissolving it with detergent. Mem-
brane proteins are difFcult to study in
NMR or X-ray crystallography, since the
use of detergents may affect the confor-
mation. So, CD spectroscopy is a useful
method for investigating the structure of
membrane proteins.
Membrane proteins tolerate mildly dis-
ruptive detergents without loss of activity.
To analyze the CD spectra of membrane
proteins requires the known structures
previous page 1390 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1392 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off