bmal1 tad  (Azenta)

Journal: Molecular cell

Article Title: A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms

doi: 10.1016/j.molcel.2017.04.011

Figure Lengend Snippet: (A) Domain schematic of mouse BMAL1 showing the chemical shifts (Δδ) in the TAD after binding CRY1 CC helix (purple) or CBP KIX domain (green). *, proline residues that lack crosspeaks in 15N-1H HSQC NMR spectra. (B) Selected regions of 15N-1H HSQC spectra of 15N BMAL1 TAD displaying backbone amide peaks for two isomers at W624 and L626. (C) Cross peaks for P623 and P625 Cβ and Cγ atoms are shown in strips from the 15N-edited (H)C(CO)NH-TOCSY of 13C/15N BMAL1 TAD at 15N planes for W624 and L626 amides. Average 13C shifts for trans (gray) and cis (blue) isomers from (Shen and Bax, 2010). (D) The W624-P625 imide bond in cis and trans conformations. (E) Sequence alignment BMAL1, BMAL2 and CYCLE from insects with a vertebrate-like clock (iBMAL1). (F) Regions of 15N-1H HSQC spectra of 8-mer switch peptides from mouse (FSDLPWPL, black) and dwarf honey bee (FSGLPWPLP, peach) showing cis and trans peaks for W624 indole. See also Figure S1.

Article Snippet: DATA AND SOFTWARE AVAILABILITY Raw data from the following 15N-1H ZZ exchange NMR time series experiments on 15N BMAL1 TAD is available at Mendeley Data: .

Techniques: Binding Assay, Sequencing

Journal: Molecular cell

Article Title: A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms

doi: 10.1016/j.molcel.2017.04.011

Figure Lengend Snippet: (A) Representation of cis content of 8-mer TAD switch peptides for P625 and W624 mutants compared to the intact 15N BMAL1 TAD. Cis content was calculated from peak volumes of residues 624 and 626 in 15N-1H HSQC and 1H-1H TOCSY NMR spectra. (B) 1H NMR spectra from FSDLPWPL (black), FSDLPWAL (red) and FSDLPWdmPL (blue) 8-mer TAD switch peptides highlighting the W624 indole region. (C) Synchronized circadian bioluminescence records from Bmal1−/−; Per2Luc mouse fibroblasts complemented with WT (gray), W624A (pink), P625A (red), or Δswitch (619X, green) Bmal1. Black line, mean luminescence ± s.d. from n = 6–8 replicates from 2 independent clonal lines in indicated colors. D) Circadian period of complemented fibroblast lines from panel (C). Individual period measurements with mean ± s.d. ***P<0.01 and ****P<0.0001 compared to WT Bmal1 by two-tailed t test. See also Figure S2.

Article Snippet: DATA AND SOFTWARE AVAILABILITY Raw data from the following 15N-1H ZZ exchange NMR time series experiments on 15N BMAL1 TAD is available at Mendeley Data: .

Techniques: Two Tailed Test

Journal: Molecular cell

Article Title: A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms

doi: 10.1016/j.molcel.2017.04.011

Figure Lengend Snippet: (A) Regions of the 15N-1H HSQC spectra showing 100 μM WT (left panels) or P625A (right panels) 15N BMAL1 TAD upon titration of CRY1 CC (purple) or CBP KIX (green), with increasing concentrations from 25–200 μM indicated by darker colors. (B) Fluorescence anisotropy data for CRY1 PHR (left) and CBP KIX (right) with wild type (black), P625A (red), P625dmP (blue) and Δswitch (green) 5,6-TAMRA-labeled short BMAL1 TAD (residues 594–626). Mean polarization data from one representative experiment (n = 4 replicates) of 3 independent assays. See also Figure S3.

Article Snippet: DATA AND SOFTWARE AVAILABILITY Raw data from the following 15N-1H ZZ exchange NMR time series experiments on 15N BMAL1 TAD is available at Mendeley Data: .

Techniques: Titration, Fluorescence, Labeling

Journal: Molecular cell

Article Title: A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms

doi: 10.1016/j.molcel.2017.04.011

Figure Lengend Snippet: Affinity of BMAL1 TAD mutants and conformationally-locked isomers for transcriptional regulators

Article Snippet: DATA AND SOFTWARE AVAILABILITY Raw data from the following 15N-1H ZZ exchange NMR time series experiments on 15N BMAL1 TAD is available at Mendeley Data: .

Techniques:

Journal: Molecular cell

Article Title: A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms

doi: 10.1016/j.molcel.2017.04.011

Figure Lengend Snippet: (A) Highlighted region of the 2D spectrum from a 15N-1H ZZ-exchange assay performed at 25°C of 15N BMAL1 TAD displaying cis (dark green), trans (dark blue), cis to trans (light green) and trans to cis (light blue) cross peaks for L626 at a delay = 1 s. Dashed circles, location of exchange cross peaks. (B) Overlay of 15N-1H HSQC spectra showing the cis and trans peaks of L626 at increasing temperatures. (C) Snapshot of ZZ-exchange assays at delay = 1 s at indicated temperatures. (D) Eyring analysis of exchange rates vs. temperature from ZZ-exchange assays with errors displayed in s.d. (E) Free energy plot showing the calculated activation energy of isomerization for the BMAL1 TAD. See also Figure S4.

Article Snippet: DATA AND SOFTWARE AVAILABILITY Raw data from the following 15N-1H ZZ exchange NMR time series experiments on 15N BMAL1 TAD is available at Mendeley Data: .

Techniques: Activation Assay

Journal: Molecular cell

Article Title: A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms

doi: 10.1016/j.molcel.2017.04.011

Figure Lengend Snippet: (A) Domain schematics for cyclophilins tested against the BMAL1 TAD and relative rate enhancement compared to uncatalyzed isomerization at room temperature. (B) Highlighted region of 15N-1H ZZ-exchange spectra displaying the W624 indole of the 15N TAD with indicated cyclophilins. Spectra from a ZZ-exchange time delay series (delay = 0–1 s) are overlaid in sequentially darker colors. (C) Synchronized circadian bioluminescence records from U2OS Bmal1-dLuc fibroblasts in the presence of DMSO, Cyclosporin A (CsA), or Deltamethrin. One individual trace shown for clarity from 2 independent assays with n = 3 replicates. (D) Mean circadian period of cultures upon treatment with DMSO (black), CsA (green) or Deltamethrin (purple) ± s.d. from n = 3 replicates with 2 independent assays. (E) Comparison of mean period changes ± s.d. as a function of CsA concentration in Bmal1−/−; Per2Luc fibroblasts complemented with WT (black), P625A (red), or W624A/P625A (gray) Bmal1. ***P<0.01 compared to WT Bmal1 by two-tailed t test. See also Figure S5.

Article Snippet: DATA AND SOFTWARE AVAILABILITY Raw data from the following 15N-1H ZZ exchange NMR time series experiments on 15N BMAL1 TAD is available at Mendeley Data: .

Techniques: Relative Rate, Comparison, Concentration Assay, Two Tailed Test