Biomedical Engineering Reference
In-Depth Information
water-soluble small molecules which do not interact with the protein. In this
way the solvent is made paramagnetic and a solvent-PRE (sPRE) can be
measured.
48,50
4.3.3.1 Advantages
Measuring the relaxation time is straightforward and does not require a
complete chemical shift assignment. PRE measurements are thus amenable to
high-molecular-weight structures and otherwise difficult experimental condi-
tions (e.g., membrane proteins
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). The effect is far-reaching and thus does not
require close contact of interaction sites for successful measurements. It is
complementary to RDC data for inter-domain placement, since it is sensitive
to both translation and rotation, whereas RDC data is only sensitive to relative
orientation.
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4.3.3.2 Disadvantages
If a structure is not known a priori it is difficult to guess good positions for the
spin-label. Structure-prediction methods can be used to suggest suitable
positions.
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It can also be experimentally challenging to find suitable
conditions to attach spin-labels. PREs measure distances at large separations,
which renders them relatively insensitive to subtle coordinate errors.
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Moreover, displacement of an atom perpendicular to the interaction vector
does not change the PRE. By placing spin-labels at different sites, however, the
resolution can be greatly enhanced due to triangulation effects.
4.3.3.3 The Bottom Line
PREs are a great tool to obtain far-reaching distance restraints where other
methods fail. To obtain precise structures despite the low resolution of PREs,
they have to be combined with other experimental data or with structure-
prediction force-fields
(see previous
section). PREs
are very
popular for
elucidating the relative positions of protein domains.
4.3.4 Pseudo-Contact Chemical Shifts
Paramagnetic centres with anisotropic g-tensor component cause a contribu-
tion to the chemical shift at distant nuclei which is called a pseudo-contact
chemical shift (PCS). The PCS yields long-range distance (ca. r
23
)and
orientational restraints (up to 40
˚
from the paramagnetic centre).
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Close to
the paramagnetic centre, the PCS cannot be measured due to line broadening
caused by the PRE effect (see above). Unless the protein in question has a
natural metal-binding site, a spin-label has to be attached by a linker.
