Biomedical Engineering Reference
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can be determined experimentally is about the longest distance that can be
derived by measuring is <10 Å for “non-conducting” systems.
CW ESR spectroscopy, which is a commonly employed method in many chemical
and biochemical laboratories, has been still intensively using in structural investigation
for two last decades. Among new tendencies in CW ESR applications the following ones
can be pointed out: 1) site directed spin labeling (Feix and Klug, 1998; Hustedt and
Beth, 2000; Mchaourab and Perozo, 2000), 2) rigid incorporation of a spin label in
proteins backbone alpha carbons (McNulty and Millhauser, 2000); 3) using fast Fourier
transform deconvolution (Xiao and Shin, 2000; Steinhoff et al., 1997), 4) electron nucler
double resonance (ENDOR) of labeled enzyme active centers (Makinen, Mustafi, and
Kasa, 1998), 5) pairwise interaction spin-spin interaction on a solely-tumbling
macromolecul,
6) and measurements of depth of immersion and
location of
paramagnetic centers (Likhtenshtein, 2000; Kulikov et al, 1989).
Recently many modifications of pulse ESR have been designed that allow to
improve the distances measurement accuracy and to expand range of distance available
for ESR spectroscopy (Eaton et al., 2000; Eaton et al., 2000; Freed, 2000; (Milov et al.,
1998) Maret, 1993; and references herein). The principle advances of such the pulse
methods is the direct determination of spin-relaxation parameters which, in turn, directly
related to spin-spin interactions depending on distances.
Several pulse methods were developed for estimation distances between two slowly-
relaxing spins. In a pulse electron-electron double resonance (PELDOR) technique a
spin echo is created by a two-pulse sequence at one microwave frequency. The timing of
a pulse at a second microwave frequency is varied (Milov et al., 1998). This method is
suitable for analysis of weak dipolar interactions. 3-pulse PELDOR with all three pulses
at the same microwave frequency (“2 + 1” sequence) was proposed by Raitsimling and
his co-workers (2000). A specific feature of the “2 + 1” technique is suppression of
dipolar interaction of randomly distributed spins, which allows the selection of a dipolar
interaction between radicals. Using a 4- pulse experiments it was possible to eliminate an
inherent dead experimental deadtime that limits the magnitude of the dipolar interaction
in 2 + 1 sequence and in 3-pulse ELDOR experiments (Pannier et al., 2000).
Pulse methods were used also for measurements of distances between a slowly-
relaxing spin (SLS) and rapidly-relaxing spin (RLS). Among them are spin echo
dephasing (Eaton and Eaton, 2000), methods based on the enhancements of the SLS in
the presence of (RLS) (Eaton and Eaton, 2000; Lakshmi and Brudvig, 2000), and
selective hole burning (Dzuba and Kawamori, 1996). In the latter technique a low-power
selective 180° pulse is used to burn a hole of a slowly-relaxing spin. The rate of the hole
broadening which in certain condition depends on interspin distansc, is monitored by the
free induction decay and two-pulse echo. This technique allows to measure distances
from 25 to 50
. The spin-spin interaction can causes modulation in the out-of-phase
echo generated in experiments with spin-polarized radical pairs (Salikhov et al. 1971,
1992; Tang et al., 1994; Dzuba and Hoff, 2000). This method is especially suitable for
estimation distance (25-40 A) between ion-radical pairs created in photosystems.
Examination of the effect of distances and orientation distribution can be done with the
use the “2 + 1”, DEER ESE, and double-quantum coherence techniques (Astashkin et
al., 1998; Milov et al., 1998; Borbat and Freed, 2000; Steinhoff. et al., 1997).
Å
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