Journal: Nature Communications

Article Title: Crosstalk between regulatory elements in disordered TRPV4 N-terminus modulates lipid-dependent channel activity

doi: 10.1038/s41467-023-39808-4

Figure Lengend Snippet: a TRPV4 N-terminal constructs used for structural analyses. b – d Purified TRPV4 N-terminal constructs analyzed by Coomassie-stained SDS-PAGE b , SEC-MALS c , and CD spectroscopy d . SDS-PAGE in b comparing all constructs side by side was carried out once to evaluate sample purity and respective molecular weight. e [ 1 H, 15 N]-TROSY-HSQC NMR spectrum of 15 N-labeled TRPV4-IDR (see Supplementary Fig. for backbone assignments). f , g SAXS pair-distance-distribution f and SAXS EOM (Ensemble Optimization Method) g , both in arbitrary units (arb. units), of TRPV4 N-terminal constructs (Supplementary Fig. ). The real-space distance distribution yields a radius of gyration of R g = 3.4 nm with a maximal particle dimension of D max = 14.0 nm for the IDR, R g = 4.1 nm and D max = 19 nm for the NTD as well as R g = 2.5 nm and a D max = 11.5 nm for the ARD. Every protein exhibits levels of conformational heterogeneity and the p ( r ) profiles should be interpreted as the summed volume-fraction weighted contribution within the sample population, and not as single-particle distributions. The statistical analyses of the fit in g was carried out using the reduced χ method (one-tailed distribution) and CorMap (one-tail Schilling distribution) test methods. The determined χ and CorMap p values are indicated in the corresponding graph. h NTD ensemble refined by EOM (Ensemble Optimization Method) , . Using a chain of dummy residues for the IDR and the X-ray structure of the TRPV4 ARD ( PDB: 3W9G ) as templates, a library of 10,000 NTD structures was generated and refined against the experimental data, allowing the comparison of the fitted versus the random pool and selecting a sub-set of ensemble-states representing the experimental data. Ten IDR conformers best representing the experimental scattering profile are depicted. Source data are provided as a Source Data file.

Article Snippet: MD simulations of the ARD in solution were initiated from the G. gallus ARD crystal structure ( PDB: 3W9G ; residues 135-382).

Techniques: Construct, Purification, Staining, SDS Page, Circular Dichroism, Molecular Weight, Labeling, Single Particle, One-tailed Test, Generated, Comparison

Journal: Nature Communications

Article Title: Crosstalk between regulatory elements in disordered TRPV4 N-terminus modulates lipid-dependent channel activity

doi: 10.1038/s41467-023-39808-4

Figure Lengend Snippet: a HDX of TRPV4 NTD and its isolated subdomains. Low (blue) to high (red) HDX shown for four time points. Areas without HDX assignment are colored white. For the ARD, HDX was visualized on the available X-ray structure of the G. gallus TRPV4 ARD ( PDB: 3W9G ). The six ankyrin repeats (AR) are indicated on top of the heat map diagram. b , c Root-mean-square fluctuations (RMSF) obtained from atomistic molecular dynamics (MD) simulations of the isolated G. gallus TRPV4 ARD in solution. RMSF at 42 °C mapped onto the ARD X-ray structure ( PDB: 3W9G ) b and RMSF per residue in simulations at increasing temperatures c . For comparison, HDX profiles after 10 2 s from a are displayed in the plot background. d , e RMSF of the ARD with respect to the central TRPV4 axis obtained from 1 µs long MD simulations of the complete TRPV4 core (see also Supplementary Movie ). d Schematic depiction of the MD simulation setup. The channel principal axis (defined as z ) is indicated as a dashed vertical line. The RMSF was calculated as the square root of the variance of the motion along this axis (∆ z ). e RMSF of TRPV4 residues 134–450 comprising the ARD. The solid line represents the average RMSF from all four protomers, the light area indicates the standard error of the mean. Source data are provided as a Source Data file.

Article Snippet: MD simulations of the ARD in solution were initiated from the G. gallus ARD crystal structure ( PDB: 3W9G ; residues 135-382).

Techniques: Isolation, Residue, Comparison