Biomedical Engineering Reference
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2 H lock signal (Fig. 16.3 c). Splitting of 6 Hz is routinely observed for large-
scale preparations at 25 mg/ml, though greater splitting can be achieved at higher
nanotube concentrations.
16.5.3
Residual Dipolar Couplings Measurement
RDC measurements in solution consist of three steps: record NMR experiments
on a non-aligned sample (isotropic), record NMR data on an aligned sample
(anisotropic), and RDC calculations.
16.5.4
Isotropic Measurements
Isotropic measurements are done without DNA nanotubes. Consequently, the
protein is unaligned and remains free to tumble. Thus, NMR experiments are
recorded with the unaligned protein allowing us to measure isotropic splittings.
Many different types of dipolar interactions can be measured this way: 1 H N -
15 N( 1 D NH ), 13 C 0 - 13 C ' ( 1 D c 0 c' ), and 15 N- 13 C 0 ( 1 D NC 0 )(seereviews[ 27 , 42 ]). The
unaligned splittings correspond to isotropic scalar couplings ( J ). See Fig. 16.4 afor
an example of the 1 H- 15 N HSQC and TROSY spectra of 500
M uniformly 15 N-
labeled ubiquitin in 20 mM tris and 200 mM NaCl buffered at pH 7.5. 1 H- 15 N
isotropic scalar couplings ( J ) were obtained from 1 J NH/2, which were measured
at 600 MHz ( 1 H frequency) by interleaving a regular gradient-enhanced HSQC
and a gradient-selected TROSY, both acquired with 80 ms of 15 N evolution.
Experiments were carried out on uniformly 13 C/ 15 N-enriched ubiquitin protein, a
76-residue protein. The structure of the ubiquitin protein has been determined by
both X-ray crystallography (PDB code 1UBQ) [ 43 ] and NMR spectroscopy (PDB
code 1D3Z) [ 44 ].
16.5.5
Anisotropic Measurements
Anisotropic measurements are done in the presence of DNA nanotubes, where a
small population of the protein is transiently aligned and remains free to tumble
most of the time. Spectra of this weakly aligned sample are recorded to measure
anisotropic splittings using the same NMR experiments as the non-aligned sample.
NMR spectra are then processed and anisotropic splittings ( J C D ) measured. See
Fig. 16.4 afor 1 H- 15 N HSQC and TROSY spectra of a 500
M uniformly 13 C- and
15 N-labeled ubiquitin sample in 20 mg/mL nanotubes, 20 mM tris, and 200 mM
NaCl, buffered at pH 7.5.
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