Real-Time NMR Characterization of Structure and Dynamics in a Transiently Populated Protein Folding Intermediate

Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g., states that become transiently populated during protein folding. Here we present a strategy for obtaining sequential NMR assignments as well as atom-resolved information on structural and dynamic features within a folding intermediate of the amyloidogenic protein β2-microglobulin that has a half-lifetime of only 20 min

Interaction of Nonstructural Protein 5A of the Hepatitis C Virus with Src Homology 3 Domains Using Noncanonical Binding Sites

Src homology 3 (SH3) domains are widely known for their ability to interact with other proteins using the canonical PxxP binding motif. Besides those well-characterized interaction modes, there is an increasing number of SH3 domain-containing complexes that lack this motif. Here we characterize the interaction of SH3 domains, in particular the Bin1-SH3 domain, with the intrinsically disordered part of nonstructural protein 5A of the hepatitis C virus using noncanonical binding sites in addition to its PxxP motif. These binding regions partially overlap with regions that have previously been identified as having an increased propensity to form α-helices. Remarkably, upon interaction with the Bin1-SH3 domain, the α-helical propensity decreases and a fuzzy complex is formed

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