Circular Dichroism in Protein Analysis
65
the absorbance is zero. Otherwise, it may
be necessary to clean the cuvette and test
again. The signal-to-noise ratio depends on
many factors: it increases as
n
1
/
2
(where
n
is the number of measurements),
I
1
/
2
(where
I
is the intensity of light beam),
and
τ
1
/
2
(where
τ
is the measuring time
for each point). The CD instrument must
be calibrated regularly using a standard
sample. The most commonly used stan-
dard for visible and UV measurements is
(
+
)-10-camphorsulfonic acid (CSA).
5.3
Solvents and Temperature Effects
Theamoun
to
fsamp
lerequ
i
redfo
rCD
experiments is relatively small compared
to other absorption spectroscopic tech-
niques. Water is the most frequently
used solvent for CD measurements be-
cause of its excellent UV transparency.
Fluorinated
alcohols,
such
as
2,2,2-
trifluoroethanol
(TFE)
and
1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP), are good
solvents
as
well.
However,
secondary
structure, especially of peptides, can be
influenced by solvents, for example, tri-
fluoroethanol promotes
α
-helical confor-
mations and nonpolar solvents, such as
lipids and destabilize
α
-helices. MeCN is
a good organic solvent. Phosphate buffers
are also useful due to their low absorbance.
However, some common salts and buffer
components do signi±cantly interfere with
the far UV spectra and are best avoided.
Temperature may have a signi±cant
effect on CD spectra. When the free energy,
difference
between
two
conformations
of a molecule,
1
G
,i
sn
o
tt
o
om
u
c
h
higher than
RT
in the range of accessible
temperatures (where
R
is the universal gas
constant), a mixture of both conformers
can
be
expected
in
solution.
In
this
case, modi±cations of the experimental
conditions can lead to a change in the
measured rotational strength and can be
calculated by
R
obs
=
R
1
+
R
2
exp
(
1
G
/
RT
)
1
+
exp
(
1
G
/
RT
)
(
7
)
where
R
1
and
R
2
are
the
rotational
strengths of these two conformers. Even
when there is no a temperature-sensitive
conformational change, there are temper-
ature effects on CD spectra. For short
helices,
the
temperature
coef±cient
is
small, but it increases signi±cantly with
helical length.
5.4
Stopped-flow
Stopped-flow CD is used for kinetic mea-
surements of conformational transitions
in proteins and in protein folding studies.
It has been applied in the near UV and
the far UV. The principle of stopped-flow
is straightforward. The apparatus uses a
drive motor to mix together rapidly two
solutions, contained in separate drive sy-
ringes, into a mixing cell. The solutions
then flow into the observation cell dis-
placing the previous contents with freshly
mixed reactants. A stop syringe is used
to limit the volume of solution expended
with each experiment and abruptly stops
the flow. The flow of solution into the
stop syringe causes the plunger to move
back and trigger data collection. Then CD
spectra may be measured as a function
of time.
The primary objective of the stopped-
flow experiments is to investigate transient
changes in chiroptical properties asso-
ciated with chemical, biochemical, and
biophysical reactions, such as structural
changes undergone during protein fold-
ing on a millisecond (ms) time scale
and refolding. In many cases, secondary
previous page 1385 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1387 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off