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In addition to nonuniform labeling schemes, another approach for observing protein-
ligand NOEs in larger systems involves collecting 3D X-filtered NOESY spectra on a
uniformly labeled sample at elevated temperatures. The decreased rotational correlation
time of the system at a higher temperature is generally expected to improve the sensitivity
of the 3D X-filtered NOESY experiment. Exceptions can occur if exchange broadening
increases with increased temperature. For this approach to be applied, it will often be
necessary to increase the thermal stability of the target protein. This can be accomplished
in a number of ways, including the rational design of point mutants, [ 43, 44 ] combinatorial
mutagenesis in conjunction with stability screening, [ 45 ] deletion of flexible loops [ 46 ] and
through the use of osmolytes. [ 47, 48 ] Although not applied here, another method that could
potentially afford high-sensitivity 3D X-filtered NOESY data on large, uniformly labeled
protein-ligand complexes is encapsulation in reverse micelles dissolved in low-viscosity
fluids; [ 49, 50 ] this can greatly reduce the rotational correlation time.
We have produced kinaseX constructs with significantly enhanced thermal stability
(J. Newitt et al ., unpublished work). Using one of these stability-enhanced constructs, a
uniformly 13 C/ 15 N-labeled kinaseX sample was prepared and complexed with an inhibitor
('kinaseX inhibitor 2'). This inhibitor has a heterocyclic core different from that of kinaseX
inhibitor 1. Figure 5.20 shows a portion of a 3D X-filtered NOESY spectrum recorded at
35 °C for the complex with kinaseX inhibitor 2. The spectral region in Figure 5.20 dis-
plays NOE interactions between the protein and a resonance at 7.83 ppm ( F 3 position)
arising from the heterocyclic core. Protein assignments for some of these peaks are based
F3 = 7.83 ppm
Met
Thr
Val - A
Val - B
2.0
1.6
1.2
0.8
0.4
0.0
F1 (H) (ppm)
Figure 5.20 Portion of a 3D X-filtered NOESY spectrum of uniformly 13 C / 15 N -labeled,
stability-enhanced kinaseX in complex with kinaseX inhibitor 2. The protein and inhibitor
concentrations used were 300 μM. The F3 (inhibitor 1 H ) plane is at 7.83 ppm. Peaks with pro-
tein resonance assignments are labeled. (Note: Val-A and Val-B refer to the γ 1 and γ methyl,
respectively, of the same valine residue.) The spectrum was recorded at 35 °C, 600 MHz 1 H
frequency, using a NOESY mixing time of 100 ms on a Varian Inova spectrometer equipped
with a Cold Probe. The spectrum is aliased in the 13 C ( F2 ) dimension.
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