Journal: Analytical and bioanalytical chemistry

Article Title: Optical approaches for single cell and subcellular analysis of GPCR-G protein signaling

doi: 10.1007/s00216-019-01774-6

Figure Lengend Snippet: FRET and BRET based biosensors

Article Snippet: Therefore, appropriate FRET or BRET probes can be designed to examine intermolecular receptor-G proteins and G protein-G protein interactions, thus measuring GPCR and G protein activation in real-time [ 126 , 127 , 122 ]. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Biosensor Name Target Number of molecules Physical type Pubmed ID Addgene plasmid # Gαi3 v1 Gαi Bimolecular or other FRET 16371464 Gαi-Gβ1 Gαi Bimolecular or other FRET 14673086 Gαi-Gγ2 Gαi Unimolecular FRET 14673086 Gαi2 Sensor v2 Gαi Bimolecular or other FRET 26799488 #69624 Gαi1 Sensor v2 Gαi Bimolecular or other FRET 26799488 #69623 RLucll-117-Gαs + GFP10-GY1 Gαs Bimolecular or other BRET 27499021 Gαs Sensor Gαs Bimolecular or other FRET 16963443 Gαq Sensor (v2) Gαq Bimolecular or other FRET 21619590 Gα13 Sensor (v2) Gα13 Unimolecular FRET 29505611 #112933 HCN2-camps cAMP Unimolecular FRET 17038640 Epac1-camps cAMP Unimolecular FRET 15231839 Epac2-camps cAMP Unimolecular FRET 15231839 mICNBD-FRET cAMP Unimolecular FRET 27003291 CAMYEL cAMP Unimolecular BRET 17283075 BFP-PMCA-GFP Ca 2+ Unimolecular FRET 17901055 AKAR4 PKA Unimolecular FRET #61619 CKAR PKC Unimolecular FRET 12782683 #14860 Open in a separate window FRET and BRET based biosensors 5.1.

Techniques: Plasmid Preparation

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: ( a ) Schematic representation of the Pt Au1a constructs used in this study. ( b ) Absorption spectrum of Pt Au1a full in the dark (black) and after illumination with blue light (blue). In the dark, the typical signature of an oxidized FMN chromophore can be detected. Upon light activation, these maxima decrease and a new absorption band appears at 390 nm, indicating FMN-cysteine adduct formation. ( c ) Recovery kinetics of the absorbance at 445 nm of Pt Au1a full , Pt Au1a bZIP-LOV and Pt Au1a LOV after light-activation. The red ( Pt Au1a full ), cyan ( Pt Au1a bZIP-LOV ) and orange ( Pt Au1a LOV ) lines in the plot represent an exponential fit to the data (black squares). Measurements were performed at a protein concentration of 20 µM and time constants represent the mean of three independent measurements. LOV, light-oxygen-voltage; FMN, flavin mononucleotide. DOI: http://dx.doi.org/10.7554/eLife.11860.003

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Construct, Activation Assay, Protein Concentration

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Normalized MALS detection of Pt Au1a full ( a ), Pt Au1a bZIP-LOV ( b ) and Pt Au1a LOV ( c ) fractionated by size-exclusion chromatography in the dark (black traces) and light (blue traces). The MALS-derived molar-mass signals are shown in green (dark runs) and blue–green (light runs). Additional experiments performed for Pt Au1a full and Pt Au1a bZIP-LOV in the light at varying protein concentrations are shown in . ( d ) Quantification of the monomer-dimer equilibrium of Pt Au1a LOV in the dark by MST. Error bars represent the standard deviation of three individual experiments. MALS, multi-angle light scattering; MST, microscale thermophoresis. DOI: http://dx.doi.org/10.7554/eLife.11860.004

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Size-exclusion Chromatography, Derivative Assay, Standard Deviation, Multi-Angle Light Scattering, Microscale Thermophoresis

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Normalized MALS detection of Pt Au1a full ( a ) and Pt Au1a bZIP-LOV ( b ) fractionated by size-exclusion chromatography at protein concentrations of 200 µM (black traces) and 100 µM (dashed gray traces) in the light. The MALS-derived molar mass signals are shown in green (200 µM) and yellow–green (100 µM). Pt Au1a full and Pt Au1a bZIP-LOV were pre-incubated at 20°C under continuous blue light illumination (400 μW cm -2 at 450 nm) from a royal blue (455 nm) collimated LED lamp (Thorlabs) for 5 min. 100 μl protein solution was subjected to size-exclusion chromatography at RT on a Superdex 200 Increase 10/300 GL column (GE Healthcare, Uppsala, Sweden) equilibrated in buffer C. LED, light emitting diode; MALS, multi-angle light scattering; RT, room temperature. DOI: http://dx.doi.org/10.7554/eLife.11860.005

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Size-exclusion Chromatography, Derivative Assay, Incubation, Multi-Angle Light Scattering

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: EMSAs of Pt Au1a full under dark ( a ) and light ( b ) conditions in the presence of 50 nM dsCYC2 promoter DNA. Quantification of the gels in . EMSAs, electrophoretic mobility shift assays. DOI: http://dx.doi.org/10.7554/eLife.11860.006

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Electrophoretic Mobility Shift Assay

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: DNA binding curves of Pt Au1a full under dark (black) and light (blue) conditions obtained by quantification of the amount of free DNA in the EMSAs shown in revealed EC 50 values of 860 nM (Hill coefficient of 1.35) in the dark and 90 nM (Hill coefficient of 1.65) in the light, indicating a 9.6-fold higher affinity of Pt Au1a full to DNA in its light compared with its dark state. EMSAs, electrophoretic mobility shift assays. DOI: http://dx.doi.org/10.7554/eLife.11860.007

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: EMSAs of Pt Au1a full under dark ( a ) and light ( b ) conditions in the presence of a 24-bp DNA probe (c = 50 nM) lacking the bZIP target sequence. Under dark conditions, no binding of Pt Au1a full to the DNA probe can be detected. Upon light activation, weak binding of Pt Au1a full to the DNA probe can be detected at protein concentrations above 5–10 µM. The significantly decreased affinity of Pt Au1a full to the DNA probe lacking the TGACGT binding motif reported for bZIP transcription factors confirms sequence-specific DNA binding of Pt Au1a full (cf. ). bZIP, basic region leucine zipper; EMSAs, electrophoretic mobility shift assays. DOI: http://dx.doi.org/10.7554/eLife.11860.008

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Sequencing, Binding Assay, Activation Assay, Electrophoretic Mobility Shift Assay

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: EMSAs of Pt Au1a full in the dark in the absence and presence of 10 mM MgCl 2 in the polyacrylamide gels as well as in the reaction and running buffers. The 35-bp DNA probe used in the EMSAs was the one used for DNA binding studies of Vf Au1 in the publication by Takahashi et. al. (5'-GGAGTATCCAGCTCCGTAGC TGACGT G GCCTCTGG-3', the bZIP target sequence is underlined). The DNA probe (388 nM) was incubated in buffer D without MgCl 2 ( a ) and with MgCl 2 ( b ) with varying amounts of purified Pt Au1a full (2, 4, 7.9, 15.9, 31.8, 63.5, 125, 250, 500, 1000, 2000, 4000 nM). EMSA runs were performed as described in the methods section. In the EMSA in the absence of MgCl 2 , the formation of a Pt Au1a full -DNA complex is already observed at the lowest protein concentration of 2 nM. At protein concentrations above 250 nM, a second Pt Au1a full -DNA band appears, which was also observed in gel shift experiments performed by Takahashi et. al. and in DNA binding studies of the CREB bZIP domain . This slower migrating band most likely represents two Pt Au1a full dimers bound to the 35-bp DNA probe, indicating the ability of unspecific DNA binding of Pt Au1a full at high protein concentrations. In the EMSAs performed in the presence of MgCl 2 , the formation of a stable Pt Au1a full -DNA complex is observed at much higher protein concentrations compared with the experiments without MgCl 2 . Additionally, the slower migrating Pt Au1a full -DNA band disappeared, indicating sequence-specific DNA binding of a single Pt Au1a full dimer to the target DNA sequence. Therefore, it can be concluded that the presence of MgCl 2 is required for sequence-specific DNA binding of Pt Au1a full . bZIP, basic region leucine zipper; EMSAs, electrophoretic mobility shift assays. DOI: http://dx.doi.org/10.7554/eLife.11860.009

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Binding Assay, Sequencing, Incubation, Purification, Protein Concentration, Gel Shift, Electrophoretic Mobility Shift Assay

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: ( a ) Crystal structure of the Pt Au1a LOV dark state monomer with the N- and C-terminal A'α and Jα helices flanking the LOV core colored in light gray and dark gray, respectively. ( b ) Blue light illumination induces formation of a parallel Pt Au1a LOV dimer. ( c ) In the dark, A'α covers the hydrophobic dimerization site on the LOV β-sheet. ( d ) Illumination results in a release of A'α from the LOV β-sheet and exposes the dimerization site. The Pt Au1a LOV molecules in (c and d) are colored according to the Eisenberg hydrophobicity scale . Reddish regions correspond to high and white regions to low hydrophobicity. ( e ) The Pt Au1a LOV light state dimer colored according to differences in deuterium incorporation in the dark and light state after 10 s of labeling. Shades of red and blue correspond to regions with increased and decreased deuterium uptake in the light, respectively. A peptide map that shows the differences in relative deuteration of dark and light experiments for all time points is shown in . All evaluated peptides for Pt Au1a LOV and their individual deuteration plots are shown in . ( f ) Pt Au1a LOV dark state monomer colored according to deuterium incorporation in the dark after 10 s labeling. Elements in ( e ) and ( f ) colored in dark gray represent regions that are not covered by peptides generated by pepsin digestion. Since rapid back-exchange of the two N-terminal residues prevents precise measurement of deuterium incorporation, these residues of all peptides are shown in dark gray, if not covered by an overlapping peptide. LOV, light-oxygen-voltage. DOI: http://dx.doi.org/10.7554/eLife.11860.010

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Labeling, Generated

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Data collection and refinement statistics. DOI: http://dx.doi.org/10.7554/eLife.11860.015

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques:

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Each box reflects one peptide and contains five different colors that correspond to the differences in relative deuteration (ΔD rel ) of dark and light (ΔD rel of Pt Au1a LOV,light - Pt Au1a LOV,dark ) experiments according to the legend on the left for the incubation times of 10, 45, 180, 900 and 3600 s (bottom up). MS/MS confirmed peptides are marked with diamonds. Secondary structure elements are taken from DSSP analysis of the Pt Au1a LOV dark state crystal structure. DSSP, define secondary structure of proteins; LOV, light-oxygen-voltage; MS/MS, tandem mass spectrometry. DOI: http://dx.doi.org/10.7554/eLife.11860.013

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Incubation, Tandem Mass Spectroscopy, Mass Spectrometry

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: ( a ) Interactions between the Jα and A'α helices observed in the Pt Au1a LOV light state dimer. The two protomers are related by two-fold non-crystallographic symmetry that does not apply to the A´α helices. ( b ) Crystal lattice contacts observed for the Pt Au1a LOV light-state dimer. The N-terminus of protomer A of a symmetry related molecule (indicated by a *) interacts with elements of the light-state dimer interface, which slightly affects the relative positioning of the two protomers. LOV, light-oxygen-voltage. DOI: http://dx.doi.org/10.7554/eLife.11860.012

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques:

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: ( a ) Changes in deuterium incorporation of Pt Au1a full mapped onto the structure of the Pt Au1a LOV light state dimer and a model of the bZIP domain. ( b–d ) Deuterium uptake plots of Jα-Iβ, A'α and leucine zipper peptides with D rel plotted against the labeling time for three independent experiments. The estimated abundance distribution of individual deuterated species is shown at the bottom. ( e ) Differences in deuterium incorporation of Pt Au1a full in the dark and light state in the presence of DNA mapped onto the Pt Au1a LOV light state dimer and a model of the bZIP domain. All evaluated peptides for Pt Au1a full in the absence and presence of DNA and their individual deuteration plots are shown in and , respectively. HDX-MS, hydrogen/deuterium-exchange coupled to mass spectrometry; LOV, light-oxygen-voltage. DOI: http://dx.doi.org/10.7554/eLife.11860.016

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Labeling, Mass Spectrometry

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Each box reflects one peptide and contains five different colors that correspond to the differences in relative deuteration (ΔD rel ) between light and dark (ΔD rel of Pt Au1a full,light - Pt Au1a full,dark ) experiments according to the legend on the left for the incubation times of 10, 45, 180, 900 and 3600 s (bottom up). MS/MS confirmed peptides are marked with diamonds. Secondary structure elements are taken from DSSP analysis of the Pt Au1a LOV dark state crystal structure and PSIPRED (Psi-blast based) secondary structure prediction . DSSP, define secondary structure of proteins; MS/MS, tandem mass spectrometry. DOI: http://dx.doi.org/10.7554/eLife.11860.017

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Incubation, Tandem Mass Spectroscopy, Mass Spectrometry

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Each box reflects one peptide and contains five different colors that correspond to the differences in relative deuteration (ΔD rel ) of light (ΔD rel of Pt Au1a full,light - Pt Au1a LOV,light ) experiments according to the legend on the left for the incubation times of 10, 45, 180, 900 and 3600 s (bottom up). The deuterium exchange rates of LOV domain peptides of Pt Au1a LOV and Pt Au1a full in the light are nearly identical, confirming that the pronounced differences in deuterium exchange rates of LOV domain peptides of Pt Au1a full in the dark and light originate from an interaction of the LOV and bZIP domains in the dark. HDX, hydrogen/deuterium-exchange; LOV, light-oxygen-voltage. DOI: http://dx.doi.org/10.7554/eLife.11860.018

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Incubation

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Each box reflects one peptide and contains five different colors that indicate deuterium incorporation after 10, 45, 180, 900 and 3600 s from HDX-MS measurements performed in the dark, normalized to the number of exchangeable amides in each peptide. Data were not corrected for back-exchange. The back-exchange rates for individual peptides under the chosen experimental conditions are in the range of ~30-–35%. Blue colors indicate low deuterium incorporation and reflect stable secondary structure elements, while red colors indicate high deuterium incorporation and flexible elements. Most of the peptides within the N-terminal domain and the bZIP–LOV linker region reach their highest deuteration level already after 10 s of labeling, indicating a significant fraction of highly dynamic or unstructured elements within these regions. MS/MS confirmed peptides are marked with diamonds. Secondary structure elements are taken from DSSP analysis of the Pt Au1a LOV dark state crystal structure and PSIPRED secondary structure prediction . DSSP, define secondary structure of proteins; HDX-MS, hydrogen/deuterium-exchange coupled to mass spectrometry; MS/MS, tandem mass spectrometry. DOI: http://dx.doi.org/10.7554/eLife.11860.019

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Labeling, Tandem Mass Spectroscopy, Mass Spectrometry

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Each box reflects one peptide and contains five different colors that correspond to the differences in relative deuteration (ΔD rel ) between light and dark (ΔD rel of Pt Au1a full,light -DNA - Pt Au1a full,dark -DNA) experiments according to the legend on the left for the incubation times of 10, 45, 180, 900 and 3600 s (bottom up). DOI: http://dx.doi.org/10.7554/eLife.11860.020

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Incubation

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: Each box reflects one peptide and contains five different colors that correspond to the differences in relative deuteration (ΔD rel ) between dark (ΔD rel of Pt Au1a full,dark -DNA - Pt Au1a full,dark ) experiments according to the legend on the left for the incubation times of 10, 45, 180, 900 and 3600 s (bottom up). DNA binding of Pt Au1a full in the dark induces similar effects within the LOV domain as illumination (cf. ), which suggests DNA-induced bZIP–LOV dissociation. The subtle differences in deuterium incorporation of Jα peptides observed after 3600 s in both comparisons most likely originate from slight differences in the experimental conditions of the HDX-MS measurements and have no functional implications. MS/MS confirmed peptides are marked with diamonds. Secondary structure elements are taken from DSSP analysis of the Pt Au1a LOV dark state crystal structure. DSSP, define secondary structure of proteins; HDX-MS, hydrogen/deuterium-exchange coupled to mass spectrometry; MS/MS, tandem mass spectrometry. DOI: http://dx.doi.org/10.7554/eLife.11860.021

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Incubation, Binding Assay, Functional Assay, Tandem Mass Spectroscopy, Mass Spectrometry

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: The MALS-derived molar-mass signals are shown in green. Pt Au1a LOV and Pt Au1a bZIP interact in the dark, which is reflected by a slight decrease of the elution volume of Pt Au1a LOV and an increase of the calculated molar mass signal. LOV, light-oxygen-voltage. DOI: http://dx.doi.org/10.7554/eLife.11860.024

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Derivative Assay

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: ( a ) Scattering curves of dark-adapted Pt Au1a bZIP-LOV (c = 5 mg/ml, black dots) and Pt Au1a bZIP-LOV in the absence (c = 5 mg/ml, blue triangles) and presence of DNA (c = 6.2 mg/ml, green squares) pre-illuminated with blue light. ( b ) Guinier and ( c ) Kratky plots of the SAXS data obtained for the different Pt Au1a bZIP-LOV states. The red lines in the Guinier plot indicate regions of the fit. All linear fits fulfil the criteria of q max •· R g ≤ 1.3. ( d ) Plots of the pair distance distribution functions (p(r)) calculated using GNOM . Blue light illumination alone and in combination with the presence of DNA results in Pt Au1a bZIP-LOV elongation. ( e ) Merged SAXS data of Pt Au1a bZIP-LOV used for ab initio and rigid body reconstructions of the Pt Au1a bZIP-LOV dark state conformation. For the low q region, data from the measurements performed at a protein concentration of 5 mg/ml was merged at q = 0.13 A -1 with the high q region of data collected at 9 mg/ml. The fits for the best DAMMIN and CORAL models are shown as red and gray dashed lines, respectively. ( f ) Merged SAXS data of Pt Au1a bZIP-LOV in the presence of DNA used for DAMMIN ab initio reconstructions. The fit for the best model is shown as red dashed line. LOV, light-oxygen-voltage; SAXS, small-angle X-ray scattering. DOI: http://dx.doi.org/10.7554/eLife.11860.028

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Protein Concentration

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: ( a ) DAMMIN low-resolution envelope calculated for Pt Au1a bZIP-LOV in the dark superimposed with an atomic Pt Au1a bZIP-LOV dark state model calculated by CORAL. Modelled loops are indicated as dots. The model is colored according to the HDX-MS data obtained for Pt Au1a full in the absence of DNA and shows differences in deuterium incorporation in the dark and light state after 10 s of labeling. The results obtained from SAXS-based rigid body modeling, ab initio modeling and HDX-MS data agree perfectly and support an interaction between the LOV domain and the leucine zipper of the bZIP domain. ( b ) Shape reconstruction of the Pt Au1a bZIP-LOV -DNA complex calculated using DAMMIN. The Pt Au1a LOV light state dimer and a model of the DNA bound bZIP domain was placed in the envelope by visual inspection. HDX-MS, hydrogen/deuterium-exchange coupled to mass spectrometry; LOV, light-oxygen-voltage; SAXS, small-angle X-ray scattering. DOI: http://dx.doi.org/10.7554/eLife.11860.025 10.7554/eLife.11860.026 Figure 7—source data 1. Rg values calculated for Pt Au1a full from SAXS data DOI: http://dx.doi.org/10.7554/eLife.11860.026 10.7554/eLife.11860.027 Figure 7—source data 2. Structural parameters calculated for PtAu1a bZIP-LOV from SAXS data DOI: http://dx.doi.org/10.7554/eLife.11860.027

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Labeling, Mass Spectrometry

Journal: eLife

Article Title: Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

doi: 10.7554/eLife.11860

Figure Lengend Snippet: ( 1 ) In the dark, Pt Au1a is dimeric and the LOV and bZIP domains interact directly thus inhibiting DNA binding of Pt Au1a. 2-4: close up of the LOV domain. ( 2 ) A'α and Jα are attached to the surface of the LOV β-sheet and are highly dynamic even in the dark. ( 3 ) Illumination with blue light causes Cys287–FMN adduct formation and results in undocking of Jα from the LOV core and increases its structural dynamics. ( 4 ) Structural changes within the LOV core together with the destabilization of Jα trigger the release of A'α from the dimerization site and also increase its flexibility. ( 5 ) The LOV domain dissociates from the leucine zipper of the bZIP domain and dimerizes, which results in an increased structural dynamics of the bZIP domain and ( 6 ) increases the affinity of Pt Au1a for its target DNA sequence. The model depicted includes results from FT-IR experiments on Pt Au1a-LOV that revealed Jα-dependent A'α release from the LOV core . FT-IR, Fourier transform infrared; FMN, flavin mononucleotide; LOV, light-oxygen-voltage. DOI: http://dx.doi.org/10.7554/eLife.11860.029

Article Snippet: The pETM-11 plasmids encoding Escherichia coli codon optimized Pt Au1a full (Epoch Life Science) and Pt Au1a LOV were kindly provided by H. Janovjak, IST Austria.

Techniques: Binding Assay, Sequencing, Fourier Transform Infrared Spectroscopy