Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

296 Werner et al.
Table 2
Models of Internal Dynamics for the Analysis of 15 N Relaxation Data
Model Optimized parameters Values of fixed parameters
1 S 2 f S 2 s = 1, τ e = 0, R ex = 0
2 S 2 f, τ e S 2 s = 1, R ex = 0
3 S 2 f, R ex S 2 s = 1, τ e = 0
4 S 2 f, τ e, R ex S 2 s = 1
5 S 2 s, S 2 f, τ e R ex = 0
chemical shifts of aromatic ring protons, which were used to monitor calcium
binding previously (42).
2. At least each series of T 1, T 2 and the pair of [ 1 H]– 15 N NOE experiments should be
acquired in a single session.
3. All pulses should be calibrated carefully and acquisition times in the indirect
( 15 N) and direct ( 1 H) dimension should be optimized for adequate resolution and
signal-to-noise.
4. A period of 10–20 min of dummy scans preceding each experiment increases the
reproducibility of peak intensities.
5. Methods to obtain quantitative information on motions in the µs to ms time-scale
include measurement of: T 2 as a function of CPMG delay (43,44), T 1ρ as a function
B 1-field strength (14,45), R 1ρ–R 1 as function of B 1-field offset (46), and T 2 at
various spectrometer field strengths (19).
6. Because resolution enhancement invariably deteriorates signal-to-noise, wellresolved
peaks may be processed with mild resolution enhancement, whereas
less well-resolved peaks may be treated with harsher window functions at the
expense of signal-to-noise.
7. Steps 2–7 of Subheading 3.2. can be performed with a number of software packages:
e.g., with a set of Felix2.3 macros, awk scripts and FORTRAN programs
developed by Dr. M. Akke and Dr. A. G. Palmer (46) (software available from the
authors at http://cpmcnet.columbia.edu/dept/gsas/biochem/labs/palmer/software.
html), or using NMRView (24).
8. There are numerous reasons why a two-parameter fit may be inadequate, such as
incorrect estimations of noise contributions using a single spectrum or the comparison
of two spectra. This can lead to unreasonably high χ 2 values and subsequent
rejection of some fits, especially for high signal-to-noise data.
9. For weak signals, reliable intensity determination can be difficult, especially for
longer delay times, which may result in apparent finite offsets. In these circumstances,
fitting the data to single exponential decays with finite offsets can lead to
statistically significant improvements of the fits.
10. Recently it has been suggested that the magnitude of the CSA may vary substantially
between residues (47,48), but this was not confirmed in a separate study
(49). In the study of cbEGF32-33 the CSA was assumed to be –170 ppm (50).