66
Circular Dichroism in Protein Analysis
structure is formed rapidly within 2 to
5 ms, and tertiary structure is formed
slowly. The two-state folding of a small
protein may occur within 3 ms. Stopped-
flow measurements require relatively few
combined shots to provide folding kinet-
ics data. It can be made over a wide
wavelength range, allowing the collection
of time-resolved CD spectra. These can
be analyzed to provide time-courses of
the development of individual secondary
structure types. However, the limitation
of stopped-flow is the ‘‘dead-time’’ of the
mixing process, which is typically more
than 2 ms. In order to study faster fold-
ing kinetics, temperature-jump methods
can be employed to shorten the observ-
ing time to 10
20 ns with laser-induced
rapid
heating
(discussed
in
Sect. 5.6).
In general, stopped-flow only provides
average
information
of
the
secondary
structure of protein; it cannot specify
precisely where the secondary structure
is formed.
5.5
Synchrotron Radiation
The X-ray source for measuring an X-
ray absorption spectrum is synchrotron
radiation. It covers all the wavelengths
of the electromagnetic spectrum with an
intensity over 100 times higher than the
conventional X-ray tubes. When charged
particles (electrons or positrons) from a
linear accelerator with a speed close to that
of light are injected into a storage ring
under high vacuum, the magnetic Felds
from the strong bending magnets around
the ring force the accelerated electrons to
follow the ring in the curved sections. The
high-energy particles hit the curved parts
and lose part of their energy as synchrotron
radiation, which is emitted tangential to
these curved sections.
Synchrotron radiation CD has been de-
veloped for more than 20 years, but it
has been used only recently in protein
secondary and tertiary structure analy-
sis. Synchrotron radiation CD provides
higher photon fluxes of linearly polar-
ized light particularly at short wavelengths
than conventional sources. There is no
need of a polarizer and the measuring
limit extends to the cut-off of the photoe-
lastic modulators (usually 120–130 nm)
with much higher signal-to-noise ratio.
So, CD spectra can be collected to a
lower wavelength limit, allowing more
accurate secondary structure content on
folding motifs and a high-intensity light
source
may
work
well
in
the
pres-
ence of salts, buffers, and detergents as
well. Collection times for synchrotron
radiation
CD
spectra
can
be
consid-
erably
shorter
than
with
conventional
instruments.
5.6
Fast Time-resolved Techniques
In order to study protein folding on short
time scales, fast time-resolved CD has been
developed. It is now possible to resolve
motions of chromophores well into the
subpicosecond time scale. Time-resolved
spectroscopy determines the spectral prop-
erties of a material at various instants
of time after a stimulus has been ap-
plied. There are two main approaches:
signal gating by sampling the output of
a fast-responding photodetector and prob-
ing with a short-duration pulse of light.
The limitations of the former are that
few detectors operate at long wavelengths
and the signal-to-noise ratio would be
unacceptable. The pump-probe technique
overcomes these limitations. It provides
high temporal resolution of repeatable,
photostimulated events.
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