Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

312 Boyd et al.
2. When studying multidomain constructs, the validity of refinement using a single
axis system vs an alignment tensor for each domain must also be justified, as
molecular alignment is expected to be affected by the rigidity of interdomain
linkages. The linkage between the two domains of the LDLR-AB pair was
assessed as rigid based on the small variation in T 1, T 2, and { 1 H– 15 N}-NOE values
throughout the central region of the construct, together with a correlation
time and anisotropy for the domain pair of 4.9 ns and 1.44, respectively, for the
structures calculated without the use of residual dipolar coupling derived
constraints. These values are in good agreement with those obtained for a
homologous pair of cbEGF domains from human fibrillin-1, 5.3 ns and 1.55,
which was found to have a rigid interdomain linkage (32).
4. Notes
1. In many cases, to provide sample stability, it has been found necessary to add
quite significant quantities of salts, leading to solutions with a high sample conductivity.
2. Guidelines are given for the preparation of samples with a total volume of 550 µL
although the use of Shigemi NMR tubes (Shigemi Co., Ltd, Tokyo, Japan) will
enable smaller volumes containing less total protein to be used.
3. Although these particular DMPC:DHPC bicelle preparations are only stable
above pH 6.0, other phospholipids, containing ether rather than ester linkages
(33), have successfully been used at lower pH.
4. If a bicelle solution does not orient initially at high temperature, repeated cooling
and warming of the sample may improve its behavior.
5. If the datasets are recorded from a spectrometer that uses the highest currently
available magnetic field strength (> = 17.6 T) then the intensity of the upfield low
frequency component of a 15N 1JNH doublet can be significantly reduced compared
to the downfield high frequency component. This is caused by cross-correlated
relaxation between the 15N– 1H dipolar and 15N chemical shift anisotropy
relaxation mechanisms. The differential line broadening is expected to increase
as the magnetic field strength, the molecular weight, or rotational diffusion anisotropy
increase. In these situations it is still possible to record two distinct 15N– 1H spectra to measure the residual dipolar coupling. In the first experiment, the
downfield low frequency narrow component can be selected using TROSY
(34,35), and for the second, a HSQC dataset is recorded (26). The residual dipolar
coupling can be measured from the difference between the 15N chemical shifts
in the HSQC and TROSY datasets, recorded for the isotropic and oriented phases.
In this case, the contributions to the 15N resonance frequency from the chemical
shift anisotropy and the dynamic frequency shifts should cancel.
6. In some cases, when the molecular alignment is fairly large, the peaks in the
spectrum from the oriented phase may be weak or missing. Ignoring relaxation
effects this may occur whenever the dipolar couplings become significant compared
to the value for the 1JNH coupling in a protein of about –92 Hz. For a HSQC