Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

306 Boyd et al.
or significant peak broadening may indicate that there is a strong interaction
between the protein and the bicelles.
3.3. Residual Dipolar Coupling Data Acquisition and Processing
1. NMR pulse sequences. Two RF pulse schemes have been described that have
proved to be suitable for measuring 15N– 1H residual dipolar couplings (29,30). In
the first of these methods, the residual dipolar couplings are measured from the
1JNH splitting in the 15N dimension of a HSQC type experiment incorporating a
S3E pulse sequence element, which is used to select for either the upfield or
downfield 15N multiplet component in a separate experiment. For the second pulse
sequence, known as IPAP, two separate 2-D datasets are recorded, corresponding
either to a J-coupled in-phase or an antiphase HSQC-type spectrum for the indirect
dimension. These datasets are then combined, by addition or subtraction, to
form two separate spectra, corresponding to the upfield or the downfield 15N multiplet component (see Note 5).
2. Parameters. Guidelines for data acquisition and processing are suggested based
on the experiments recorded on the LDLR-AB module pair. In this study, the first
of the pulse sequences aforementioned was used where (t 15
1 N, t2
1H). 128 × 2048
complex points were acquired, with acquisition times of 64 and 82 ms, respectively.
The data were processed to give a final digital resolution of 0.98 Hz/pt (F1) and 3.05 Hz/pt (F2). Pairs of spectra were collected at 24°C and 39°C.
3.4. Data Analysis
1. Residual dipolar couplings 1 D NH in units of Hz are extracted from the difference
in 1 J HN splitting observed for the datasets recorded in the aligned (39°C) and
isotropic (24°C) phases, as illustrated in Fig. 2 (see Note 6). In the analysis, we
assume that the only dipolar couplings contributing to the experimental 1 J NH splitting
are from the directly attached amide proton (see Note 7).
2. In order to use residual dipolar couplings as restraints in structure refinement, it
is necessary to relate these values to the orientation of the molecular alignment
tensor A. For this purpose the relationship describing the dipolar coupling 1 D NH,
equation 1, may be conveniently recast,
1DNH(θ,φ) = Da {(3cos2 θ – 1) + 1.5R(sin2 θ cos 2φ)} (2)
where Da in Hz and Dr are the axial and rhombic components of the alignment
tensor
Da = 1/3[Dzz – (Dxx + Dyy)/2] and Dr = 1/3[Dxx – Dyy] (3)
R, the rhombicity defined as D r/D a, is always positive and employing this definition
will vary between 0 and 2/3 (8), and D a = (1/2 S NHγ Nγ H h [µo/8π 2 ])Azz
is the axial component of the alignment tensor A. It has been recommended that
residual dipolar coupling restraints should only be implemented for residues in
well-ordered regions of the molecule, where S 2 > 0.8. In the ideal case, with many
residual dipolar couplings sampling a wide range of orientations with respect to