Journal: Nature Communications

Article Title: Structural insights into substrate recognition by the SOCS2 E3 ubiquitin ligase

doi: 10.1038/s41467-019-10190-4

Figure Lengend Snippet: Position and characterization of SNPs of SOCS2. a Sequence conservation mapped onto the SH2 domain of SOCS2 (shown as surface). Conservation surface representation based upon the ClustalW multiple sequence alignment of CIS and SOCS1–SOCS7 sequences where highly conserved residues are shown in red/orange color and variable residue positions colored white/gold. Positions of SNP mutations are highlighted. b Transverse relaxation rates ( R 2 ) of spy molecule 3 binding to SBC protein variants. The R 2 relaxation rates were obtained by fitting as exponential decay the 19 F peak integrals of 3 measured at five CPMG delays in a sample in the absence or presence of SBC protein (see Supplementary Fig. ). Error bars reflect the quality of the fit between the non-linear least-square curve and the experimental data

Article Snippet: The T 2 relaxation time was calculated by fitting the data (Supplementary Fig. 7) as a mono exponential decay (GraphPad Prism 6) using the equation below 5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$I\left( t \right) = I\left( 0 \right)e^{ - t/T_2},$$\end{document} I t = I 0 e - t ∕ T 2 , where I ( t ) is the signal intensity or integral at CPMG filter t (in seconds), I (0) is the signal intensity when t = 0, and T 2 is the time constant of decay.

Techniques: Sequencing, Residue, Binding Assay

Journal: eLife

Article Title: Auxiliary subunits keep AMPA receptors compact during activation and desensitization

doi: 10.7554/eLife.40548

Figure Lengend Snippet: ( A ) Control recording of wild-type (WT) GluA2 receptors in response to a trapping protocol. Movements of the piezo reflecting the solution exchange are shown in thin, black lines above the current trace. Composition of the solutions is indicated in thick lines above the piezo trace. Downward ticks are 200 ms control jumps from DTT (1 mM) to DTT and glutamate (Glu, 10 mM). Four pre-trap control pulses were followed by a 1 min long trapping pulse (red line) in Glu (10 mM) and M3M (1 μM). After the trapping pulse, the patch was exposed to 10 post-trap control pulses. The first post-trap pulse gives no response because receptors are desensitized (for details, see Materials and methods). ( B ) Same as in ( A ), but for V666C mutant. The top two recordings are from the same patch: in the first trace, V666C receptors were exposed only to Glu. Trapping of the same patch in Glu and M1M (1 μM), results in pronounced peak current reduction, which partly recovered with τ recovery = 30 ± 7 s, n = 5 (grey dots; post-trap control pulses extended to 30 for fit). The bottom trace is a different patch trapped in bMTSp (1 μ M ), showing even stronger peak current reduction without any recovery. ( C ) As in ( B ) but for the A665C mutant. The two traces are paired recordings of the same patch. Post-trap control pulses show that A665C cross-links to itself, but most of the current recovers within several seconds after the trap (grey dots; τ recovery = 3.3 ± 0.4 s, n = 17). If the same patch is now trapped in Glu and M8M (1 μM), the peak current reduction is much more pronounced and does not recover. ( D ) Summary of the trapping effects for WT (black) and V666C (red) for cross-linkers M1M (7 Å) to M10M (18 Å). Trapping effect after 1 min was calculated as the ratio of the post-trap and pre-trap peak current (arrows in A-C). MTSEA is a monofunctional reagent and ‘w/o MTS’ stands for ‘without MTS’ (Glu only, pooled for all experiments). Dashed line indicates no effect. For peak current reduction in a bis-MTS vs. w/o MTS (pooled), p < 10 −7 , for all cross-linkers. ( E ) Same as in ( D ), but for A665C (red) in cross-linkers M3M (9 Å) to M10M (18 Å). A665C mutant in the presence and absence of an MTS cross-linker resulted in p ≤ 0.02, depending on the cross-linker. For statistics vs. WT and between different bis-MTS, see . ( F ) Trapping time for V666C receptors in M3M, M6M and M10M. The 1-min trapping protocol was repeated up to six times, resulting in a cumulative exposure of the patch to a bis-MTS of up to 6 min. The data were fit with a monoexponential for each cross-linker (τ indicated in brackets). ( G ) Trapping profile of desensitized V666C receptors shows the effect of each cross-linker vs. its length, in the first minute of exposure. The data were fit with a parabola (red line): f ( x ) = K 0 + K 1 *( x – K 2 ) 2 (for details, see Materials and methods). 10.7554/eLife.40548.008 Figure 2—source data 1. Statistics of trapping desensitized wild-type (WT), A665C and V666C receptors with different bis-MTS cross-linkers; data from . The effect was measured after 1 min of exposure to a given bis-MTS reagent.

Article Snippet: The resulting current decay was described by a monoexponential fit in Igor Pro (v7.06, Wavemetrics, Lake Oswego, Oregon, USA, RRID: SCR_000325 ).

Techniques: Control, Mutagenesis

Journal: eLife

Article Title: Auxiliary subunits keep AMPA receptors compact during activation and desensitization

doi: 10.7554/eLife.40548

Figure Lengend Snippet: ( A ) Trapping protocol for V666C receptors in 50 μM M3M and 10 mM glutamate (Glu), same as in , but with a trapping jump consisting of 50 ms-pulses into 50 μM M3M and 10 mM Glu at 6.7 Hz (thick, red lines), interspersed with 100 ms intervals in Ringer solution. ( B ) Progression of the peak current reduction in response to the trapping pulses (red in ( A )) was determined for each patch, normalized to the first pulse, averaged across patches and plotted. The resulting decrease in the peak current was fit with a monoexponential (red line) with τ = 0.2 s. Dashed line is a fit to trapping in 1 μM M3M from . ( C ) As in ( A ), but for M10M, with the trapping jump prolonged to 1 min to complete the peak current reduction. ( D ) As in ( B ), but for M10M. Peak current decay was best fit with a double exponential resulting in weighted τ W of 1.7 s.

Article Snippet: The resulting current decay was described by a monoexponential fit in Igor Pro (v7.06, Wavemetrics, Lake Oswego, Oregon, USA, RRID: SCR_000325 ).

Techniques:

Journal: eLife

Article Title: Auxiliary subunits keep AMPA receptors compact during activation and desensitization

doi: 10.7554/eLife.40548

Figure Lengend Snippet: ( A ) To check whether M10M modifies active V666C receptors silently, a patch containing V666C receptors was first exposed to Glu (10 mM) and M10M (1 µM) in CTZ (100 µM) to block desensitization as shown in (1). Colors are as in and . The patch was then exposed to short (100 ms) jumps into 10 mM Glu (in reducing conditions), until CTZ unbound, as deduced from desensitization of the response (2). Freely desensitizing receptors in the same patch were then exposed again to M10M and glutamate, without CTZ (3). ( B ) Summary of effects on V666C receptors in active state (M10M + CTZ) and after CTZ unbinding (M10M-CTZ) (paired recordings). For comparison, the trapping of patches that were never treated with CTZ (M10M) is also plotted. Pre- and post-trap current amplitudes were measured from peaks indicated with arrows in ( A ). P value compares patches from which CTZ unbound and patches that were never treated with CTZ. ( C ) Time constant of deactivation (τ OFF ) was measured for active V666C receptors before (blue) and after (red) 1 min trap in M10M (from control pulses as indicated with a blue and red star in (A-1)). Normalized current decays were fit with a monoexponential. ( D ) Summary of τ OFF as determined in ( C ).

Article Snippet: The resulting current decay was described by a monoexponential fit in Igor Pro (v7.06, Wavemetrics, Lake Oswego, Oregon, USA, RRID: SCR_000325 ).

Techniques: Blocking Assay, Comparison, Control

Journal: eLife

Article Title: Auxiliary subunits keep AMPA receptors compact during activation and desensitization

doi: 10.7554/eLife.40548

Figure Lengend Snippet: ( A ) Trapping speed of M6M for active V666C receptors (empty circles) and desensitized in the presence (black diamonds) and absence (black circles) of Stargazin (Stg). Receptors were exposed to 1 μM M6M for 1 min intervals (as described and Materials and methods), with up to six sequential applications. Current reduction was determined after each 1-min exposure, averaged across patches and plotted here vs. cumulative trapping time. Current decay was monoexponential in every case, with τ indicated in brackets. ( B ) Same as in ( A ), but for M10M. ( C ) Trapping speed of M1M for active V666C receptors in the presence (black diamonds) and absence (empty circles) of Stg. ( D ) Same as in ( C ) but for M8M.

Article Snippet: The resulting current decay was described by a monoexponential fit in Igor Pro (v7.06, Wavemetrics, Lake Oswego, Oregon, USA, RRID: SCR_000325 ).

Techniques: