Biomedical Engineering Reference
In-Depth Information
difference in chemical shifts between a diamagnetic and paramagnetic sample.
With as few as 20 measured PCS, the remaining PCS can be predicted to aid in
resonance assignment.
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6.2.3 Applications for Studying Lowly Populated States
PCS and RDCs can be used as restraints for structure refinement and
determining domain motion within a protein or a complex. This has been done
by several groups for the two-domain protein calmodulin (CaM). In 1999,
Biekofsky et al. loaded paramagnetic terbium (Tb
3+
) into one of the two N-
terminal calcium binding sites of CaM while calcium remained bound to the C-
terminal binding site. Smaller than expected PCS and RDC values were
observed for the free protein compared to when bound to a target peptide. This
suggested that the free protein occupied several conformations in solution.
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This work was continued by Bertini et al. in 2004 using Tb
3+
and thulium
(Tm
3+
). The conformation space sampled by CaM was determined and it was
found that neither the fully extended nor the closed conformation was favored
in solution. The orientation of the C-terminal domain was also mapped and it
was found to occupy a large elliptical cone with an axis tilted y30u relative to
the N-terminal binding site.
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PCS were also used to study the transient photosynthetic plastocyanin-
cytochrome f complex. In 1998, Ubbink et al. published a method to determine
the orientation of plastocyanin in the complex based on intermolecular PCSof
the plastocyanin nuclei caused by the cytochrome heme iron.
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In similar studies of this complex from other species it was found that the PCS
were not in agreement with a single, well-defined structure. The data suggested
averaging of the PCS due to mobility of plastocyanin within the complex.
41,75
In
2008, Hulsker et al. used random rotation modelling to generate an ensemble of
plastocyanin orientations that was in agreement with the experimental data
(Figure 6.1).
41
It is clear that plastocyanin has considerable freedom to sample
the surface of cytochrome f within the complex.
6.3
Paramagnetic Relaxation Enhancement
6.3.1 PRE Theory
Structural information about lowly populated states can be obtained using
PRE, which measures the increase in the transverse relaxation rate of the
minor state (C
LP
2
) caused by a nearby paramagnetic center. This technique
requires that the minor state experiences larger PRE than the dominant state,
generally due to the minor state being closer to the paramagnetic center. It also
requires that exchange between the states occurs much faster than the PRE
rate of the minor state (k
ex
ยป C
LP
2
). When these conditions are met, structural
restraints for ensemble modelling can be obtained from the PRE profile.
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