Figure 1.
Cell-free expressed cytosolic constructs of the T cell- and B cell receptor.
(A) Cartoon of the receptors with indicated immunoreceptor tyrosine-based activation motifs (ITAMs), (B) SDS-PAGE gel of in vitro expressed disordered constructs in levels suitable for NMR experiments.
Figure 2.
Backbone assignment for CD79a.
(A) Progress of the assignment during the course of TA procedure for CD79a samples with concentrations of 330, 120, 60, and 30 µM. The horizontal axis shows the total measurement time excluding the HNCO experiment, which was recorded prior to the TA. The spectral processing and automated analysis were always shorter than the data acquisition and, thus, can be performed in real time; (B) assignment validation for 60 µM (blue bars) and 30 µM (red bars) samples. The assignment probabilities are plotted for the most probable and alternative assignments (shaded and solid bars, respectively). The probability scores were calculated as a fraction of successful assignments over 30 resample trials.
Figure 3.
Progress of targeted acquisition versus total measurement time for a 120 µM sample of CD79a.
Build-ups are shown for the number of assigned residues and the number of detected peaks in individual BEST-TROSY-type experiments [14].
Table 1.
Samples produced using CFPS and backbone assignments obtained with the automated TA procedure.
Figure 4.
Zoomed region of the 15N-HSQC of CD79a in different conditions.
(A) NaPi buffer, (B) 6 M urea, (C) 20% TFE, (D) reduced spin label (MTSL) attached to CD79a, (E) K4C/C35S form, (F) Y25E/Y36E form. Selected peaks are annotated to show rearrangements of the signal position for the different conditions (19Y, 35M) or position of the specific mutations (4K, 33C, 25Y, 36Y). Peaks outside the zoomed region are shown as arrows pointing towards the correct position.
Figure 5.
13C secondary chemical shifts for CD79a in native (red) and 20% TFE (blue) states.
The values are obtained as the difference between the observed and random coil chemical shifts. The latter are measured in 6 M urea solution. The secondary chemical shifts of CO (dashed lines), CA (bold lines) and CB shifts (thin lines) clearly indicate α-helix propensities in the ITAM-motif region shown by horizontal black bars.
Figure 6.
Assignment validation procedure with jackknife resampling.
See text for explanations.