Journal: bioRxiv

Article Title: Cryo-EM structure of the volume-regulated anion channel LRRC8

doi: 10.1101/331207

Figure Lengend Snippet: ( a ) Close-up view of the neighboring EL1 helices forming the pore constriction site. The side chains are depicted by stick models. ( b ) Sequence alignment around the pore constriction site, depicted according to Extended Data Fig. 2. ( c ) VRAC activity in the LRRC8A-rescued LRRC8A -knockout HEK293A cells. Each line indicates the mean ± s.e.m. of the normalized YFP fluorescence ( n = 4). Hypotonicity after the addition of iodide: 231 mOsm. WT (+) and WT (++++) represent the different expression levels of the LRRC8A wild-type protein detected by western blotting, according to Extended Data Fig. 6c. ( d ) Maximum speed of hypotonicity-induced YFP quenching. Data are presented as the mean ± s.e.m. with each individual light grey point (n = 4). ** P < 0.01, *** P < 0.001 by Dunnett’s test.

Article Snippet: The human LRRC8A WT protein (NCBI Reference sequence number: NP_062540.2) was cloned from human brain cDNA (ZYAGEN) into the pEG BacMam vector, with an C-terminal GFP-His8 tag and a tobacco etch virus (TEV) cleavage site, and was expressed in HEK293S GnTI − (N-acetylglucosaminyl-transferase I-negative) cells (ATCC, cat. no. CRL-3022) .

Techniques: Sequencing, Activity Assay, Knock-Out, Fluorescence, Expressing, Western Blot

Journal: bioRxiv

Article Title: Cryo-EM structure of the volume-regulated anion channel LRRC8

doi: 10.1101/331207

Figure Lengend Snippet: ( a ) Subcellular localization of LRRC8A mutants rescued in LRRC8A -knockout HEK293A cells. The white dashed square indicates a region around the cell membrane; the magnified image of the LRRC8A-derived fluorescent channel is located in the corner. The white scale bar indicates 25 μm. ( b ) FSEC profiles on a Superose 6 Increase 10/300 GL column (GE Healthcare) for the EGFP-fused HsLRRC8A mutants expressed in HEK293S GnTI − cells. The arrows indicate the estimated elution positions of the void volume, the EGFP-fused HsLRRC8A, and the free EGFP. The analysis of the mutants showed symmetric and monodisperse peaks, similar to those of the wild-type channel, confirming the proper structural integrities of these mutant channels. ( c ) Amounts of HsLRRC8A expression in rescued LRRC8A -knockout HEK293A cells. † indicates non-specific bands. ( d ) Comparison between individual time courses and the fitting curve of the normalized YFP fluorescence. Grey lines and light blue lines indicate individual time courses of normalized YFP fluorescence ( n = 4). Blue lines indicate the mean of one-phase exponential decay-fitting curves. I − -iso: 332 mOsm, I − -hypo: 231 mOsm.

Article Snippet: The human LRRC8A WT protein (NCBI Reference sequence number: NP_062540.2) was cloned from human brain cDNA (ZYAGEN) into the pEG BacMam vector, with an C-terminal GFP-His8 tag and a tobacco etch virus (TEV) cleavage site, and was expressed in HEK293S GnTI − (N-acetylglucosaminyl-transferase I-negative) cells (ATCC, cat. no. CRL-3022) .

Techniques: Knock-Out, Derivative Assay, Mutagenesis, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Cryo-EM structure of the volume-regulated anion channel LRRC8

doi: 10.1101/331207

Figure Lengend Snippet: ( a ) The conservation score mapped on the molecular surface of HsLRRC8A. The score was calculated by the program CONSURF , based on the multiple-sequence alignment of the A-E isomers in . The molecular surface is colored according to the normalized conservation score, from 0.752 (pink) to −0.983 (purple). ( b ) The model structure of the (LRRC8A) 5 ·LRRC8D heterohexamer, viewed parallel to the membrane (left), and from the extracellular side (right). The homology model of human LRRC8D was generated by the program MODELLER , using the alignment in , and then the α subunit of the present structure was replaced by this model. In the inset, a close-up view of the channel pore forming regions from the extracellular side is shown, and the side chains of pore-lining amino residues are depicted in stick representations. The five LRRC8A subunits are colored light blue, while the LRRC8D isoform is colored light brown.

Article Snippet: The human LRRC8A WT protein (NCBI Reference sequence number: NP_062540.2) was cloned from human brain cDNA (ZYAGEN) into the pEG BacMam vector, with an C-terminal GFP-His8 tag and a tobacco etch virus (TEV) cleavage site, and was expressed in HEK293S GnTI − (N-acetylglucosaminyl-transferase I-negative) cells (ATCC, cat. no. CRL-3022) .

Techniques: Sequencing, Generated

Journal: ACS Synthetic Biology

Article Title: Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas

doi: 10.1021/acssynbio.0c00082

Figure Lengend Snippet: Riboswitch regulation of casbene production. Casbene production was achieved by introducing the casbene synthase expression cassette into the nuclear genome of the Chlamydomonas UVM4 strain. (a) Schematic of the expression cassette shows the contributing parts assembled in the following order, HSP70/RBCS2 promoter, 22 nt RBCS2 5′UTR, CrTHI4–4N RS , PSAD chloroplast target peptide (cTP shown as blue box), codon optimized casbene synthase ( CBS ) containing multiple copies of the Chlamydomonas RBCS2 intron 1 (i1) fused with a GS linker peptide (orange box) to Venus containing RBCS2 intron 2 (i2), the 3′ UTR was derived from CA1. (b) Casbene production in a Chlamydomonas transformant that expressed the casbene synthase transgene was assessed using Gas Chromatography Mass Spectrometry (GC-MS). A representative transformant was cultured in TAP media with a 10% n-dodecane overlay. Nine days postinoculation, the overlay was analyzed by GC-MS. Casbene captured by the n -dodecane overlay was detected at the expected retention time (black trace), thereby confirming that the casbene synthase enzyme fused to Venus fluorescent protein was functional. The GC-MS ion chromatogram ( m / z 121) shows metabolites carrying a mass-to-charge ratio ( m / z ) of 121 ± 0.5. Internal standard β-caryophyllene was detected at 12.32 min retention time (RT), while casbene was detected at 23.16 min RT. In agreement with previous reports, a selection of oxidized casbene molecules was also detectable between RT 25.6 and 26.7 RT. The detection of casbene and its oxidized derivatives demonstrates capacity of this transformant to produce casbene. When this transformant was cultured in media containing 10 μM thiamine (green trace), casbene was not detected, a result that highlighted the utility of the riboswitch for regulation of transgene expression.

Article Snippet: The amino acid sequences of the Jatropha curcas casbene synthase (JcCBS, NCBI Reference Sequence: NP_001292945.1) was codon optimized for expression from the Chlamydomonas nuclear genome and synthesized by GenScript Corporation (USA).

Techniques: Expressing, Derivative Assay, Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Cell Culture, Functional Assay, Selection

Journal: Biochemistry and Biophysics Reports

Article Title: Expression and purification of the full murine NPM2 and study of its interaction with protamines and histones

doi: 10.1016/j.bbrep.2016.04.002

Figure Lengend Snippet: Native PAGE of a titration of histone octamers (A) and mouse protamines P1/P2 (B) with increasing amounts of M.NPM2. ). In this type of analysis, NPM2: histone/protamine complexes display a complex ‘shift’ and are unable to enter the gel. (C) Amino acid sequence of mouse NPM2 and core histones H2A, H2B and H4, with interaction sites determined through CXMS data ( a) highlighted in red. Three peptide sequences in NPM2 represent strong cross-linking candidates with sequences in H2A, H2B and H4 (highlighted in red). Gallus gallus H2A NCBI Reference Sequence: AAC60008.1, G. gallus H2B NCBI Reference Sequence: AAC60000.1 , G. gallus H4 NCBI Reference Sequence: NP_001032932.1. (D) Amino acid sequence of mouse NPM2, P1 and P2, with possible interaction sites determined through CXMS data ( a) highlighted in red. Three peptide sequences within M.NPM2 represent possible cross-linking candidates. M. musculus NPM2 NCBI Reference Sequence: NP_851990.2; M. musculus P1 NCBI Reference Sequence: NP_038665.1; M. musculus P2 NCBI Reference Sequence: P07978.1.

Article Snippet: Gallus gallus H2A NCBI Reference Sequence: AAC60008.1, G. gallus H2B NCBI Reference Sequence: AAC60000.1 , G. gallus H4 NCBI Reference Sequence: NP_001032932.1. (D) Amino acid sequence of mouse NPM2, P1 and P2, with possible interaction sites determined through CXMS data ( a) highlighted in red.

Techniques: Clear Native PAGE, Titration, Sequencing