Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.

We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution

Modulation of Structural Heterogeneity Controls Phytochrome Photoswitching

Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although crystal structures of dark and light adapted states have been determined, the molecular mechanisms underlying photoactivation remains elusive. Here we demonstrate that the conserved tongue region of the PHY domain of a 57kDa photosensory module of Deinococcus radiodurans phytochrome, changes from a structurally heterogeneous dark state to an ordered light activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.peerReviewe

Zoomed region of the ¹⁵N-HSQC of CD79a in different conditions.

<p>(<b>A</b>) NaPi buffer, (<b>B</b>) 6 M urea, (<b>C</b>) 20% TFE, (<b>D</b>) reduced spin label (MTSL) attached to CD79a, (<b>E</b>) K4C/C35S form, (<b>F</b>) 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.</p

Cell-free expressed cytosolic constructs of the T cell- and B cell receptor.

<p>(<b>A</b>) Cartoon of the receptors with indicated immunoreceptor tyrosine-based activation motifs (ITAMs), (<b>B</b>) SDS-PAGE gel of <i>in vitro</i> expressed disordered constructs in levels suitable for NMR experiments.</p

Assignment validation procedure with jackknife resampling.

<p>See text for explanations.</p

Progress of targeted acquisition versus total measurement time for a 120 µM sample of CD79a.

<p>Build-ups are shown for the number of assigned residues and the number of detected peaks in individual BEST-TROSY-type experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062947#pone.0062947-Favier1" target="_blank">[14]</a>.</p