Biomedical Engineering Reference
In-Depth Information
where h is defined as tan
21
(v
1
/V). Changing the frequency, V, or amplitude,
v
1
, of the spin-lock pulse alters the effective strength of the spin-lock field,
v
e
~(v
1
zV
2
)
2
. This attenuates R
ex
in a way similar to changing the number of
refocusing pulses in the CPMG experiment. However, compared to CPMG
based experiments, higher effective rf fields can be achieved and therefore
RFRD experiments are more sensitive to faster motions. Structural and kinetic
information can be obtained by fitting R
2,eff
versus effective field strength to an
exchange model.
113
6.4.2 Applications for Studying Lowly Populated States
In 2007, Wang et al. and Eichm ¨ ller and Skrynnikov exploited paramagnetic
centers for the study of protein dynamics. Wang et al. substituted a
paramagnetic Co
2+
ion into the Zn
2+
binding site of the N-terminal domain
of PA0128, a protein of unknown function from Pseudomonas aeruginosa.
CPMG RD was used to study chemical exchange, which was attributed
primarily to interactions between the N-terminal domain and a single b-strand
of the C-terminal domain. This study demonstrated that PCS induced by
paramagnetic centers could enhance RD effects while also providing structural
information.
71
At the same time, Eichm ¨ ller and Skrynnikov used R
1r
spin-lock pulses to
investigate the dynamics of cardiac troponin, in which La
3+
,Ce
3+
or Pr
3+
ions
occupied the native Ca
2+
binding site. It was shown that PCS induced by the
lanthanide ions made it possible to measure long-range dispersion effects,
particularly when the local environment of the nuclei is unchanged such as in a
rigid a-helix structure. However, the authors pointed out the need for generally
applicable lanthanide-binding tags and they performed preliminary testsof
Ca
2+
-bound cardiac troponin with lanthanides chelated to MTS-EDTA tags.
However, the length and flexibility of the tags resulted in substantial averaging
of the paramagnetic effects.
107
More promising results were obtained for rigid, two-point attached
lanthanide-binding tags by Hass et al. in 2010. CLaNP-5 tags loaded with
Lu
3+
,Yb
3+
or Tm
3+
were bound via double cysteine mutations to an a-helix
and a loop region of pseudoazurin as well as a b-strand of Cc. CPMG RD was
used to study local protein dynamics and the effects were found to be highly
dependent on the attachment site within the protein. Large RD effects were
observed when the tag was bound to Cc or the loop region of pseudoazurin,
which is known to be dynamic, but only moderate effects were observed when
bound to the a-helix of pseudoazurin. This highlighted the importance of rigid
tag attachment and affixing tags at multiple positions may be required to
isolate the dynamics of the tag from those of the protein.
34
Nevertheless, the
combination of paramagnetic lanthanide tags with RD provides a promising
approach for studying dynamics of lowly populated states, due to the strong
RD effects caused by the PCS changes (Figure 6.3).
