Circular Dichroism in Protein Analysis
67
Time-resolved CD can provide infor-
mation on structural rearrangement and
folding for both smaller chiral species and
large macromolecules in different envi-
ronments. Conventional time-resolved CD
spectroscopy is limited by the time scale
of the polarization modulator employed.
Now, with shorter-pulse He–Ne lasers and
a photoelastic modulator to alternate the
pump laser polarizations without limiting
thet
imeresponseofthemeasuremen
t
,it
is possible to detect the inherently weak
difference between two large absorption
signals, which are ellipsometric and are
subject to signiFcant optical artifacts. The
limitation is the pulse length of the grat-
ing forming (pump) laser. Measurements
canbemadeonmicrosecond,nanosecond,
and picoseconds timescales. Although pi-
cosecond resolution is achievable, there
aresometechnicalproblems,includingin-
tensity instability, polarization scrambling,
and tunability limitations.
6
Spectral Characteristics of Elements of
Protein Structures
CD is commonly used to probe the sec-
ondary conformations of proteins to study
protein folding and to investigate inter-
actions of proteins with small molecules
such as achiral molecules whose induced
CD is due to their interaction with the pro-
tein. Changes in CD can be used to provide
evidence for conformational changes and
to determine equilibrium constants. A few
examples of conformational transitions
are the
α
-helix to
β
-sheet conformational
switch in the prion protein as the concen-
tration of sodium dodecyl sulfate is altered;
the unfolding of the coiled-coil structure of
Fbrinogen binding protein as temperature
increases; the increasing helical content of
troponin-C on binding Ca
2
+
and the bind-
ing of molybdate by the protein ModE,
which gives large changes in the Trp sig-
nal at 292 nm. ±ar UV and near UV CD
can be used to monitor protein unfolding
upon the alteration of pH or the addition
of a denaturant, such as urea or guani-
dinium chloride (GdmCl). CD can also be
used to examine the interactions between
multiple domains of proteins, for example,
the flavin domain and the heme domain
of flavocytochrome P450-BM3 give char-
acteristic changes in the environment of
the cofactors when they are linked in the
intact enzyme.
6.1
α
-helix
The right-handed
α
-helix (±ig. 4) has main-
chainhydrogenbondsbetweenaminoacid
residues and positions
i
and
i
+
4a
long
the backbone chain. There is a 0.15-nm
translation and a 100
rotation between
two consecutive peptide units. Its helical
pitch (the number of residues multiplied
by the distance between
α
-carbon atoms on
neighboring residues) is 0.54 nm. The
α
-
helix has three distinctive bands in the far
Fig. 4
The main chain of a polypeptide in an
α
-helical conformation, with hydrogen bonds denoted
by dashed lines.
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