Journal: Nature

Article Title: Structure of the Insulin Receptor-Insulin Complex by Single Particle CryoEM analysis

doi: 10.1038/nature26153

Figure Lengend Snippet: Human IR ECD binding and kinetic data . Top: Human IR ECD binding data for recombinant human insulin. Data is reported as the geometric mean for 23 replicates; the geometric standard deviation is shown in parentheses. The potency measured with the 125 I-insulin competition assay agrees very well with values from the literature 17 . Bottom: Human IR ECD Binding Kinetic Data for RHI. Data is reported as the geometric mean for three replicates; the geometric standard deviation is shown in parentheses. The SPR data is very consistent with values from the literature 31 .

Article Snippet: Recombinant human Insulin Receptor/CD220 was obtained from R&D systems (catalog # 1544-IR-50/CF, https://www.rndsystems.com/products/recombinant-human-insulin-r-cd220-aa-28-944-protein-cf_1544-ir-cf ), reconstituted in PBS (Phosphate buffered saline, pH=7.4) at 0.3 mg/ml (0.00293 mM), stored in 100 ul aliquots at -80, and used as is (without further purification).

Techniques: Binding Assay, Recombinant, Standard Deviation, Competitive Binding Assay

Journal: Nature Communications

Article Title: Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

doi: 10.1038/s41467-023-44565-5

Figure Lengend Snippet: Three active-site conformations, including the central FMN (gray), Gln (Q, cyan/green/yellow), and Tyr (Y, gray), as well as the nearby Trp (W, cyan/green/yellow) in BLUF domains. W in NH in configuration ( a ) is adopted from one X-ray structure of the AppA BLUF domain (PDB: 1YRX ). A preferred no-proton relay geometry of Tyr and Gln is depicted according to previous free energy calculations . W in NH out ( b ) and W out ( c ) configurations are adopted from the two subunits of Sy PixD tetramer (PDB: 2HFO ), chain D, and chain B, respectively. Both favor a proton transfer geometry where Tyr OH is H bonded to Gln Oε1, and Gln Nε2H is H bonded to FMN N5 , . Edge-to-edge distances (double arrows) between the nearby Trp and FMN are shown in each configuration. Possible proton transfer pathways are drawn in dashed lines.

Article Snippet: The 5FW and 7FW labeled protein samples, i.e., WT and YnF mutants of Oa PAC 1-102 , Sy PixD 1-150 , and AppA 1-124 BLUF domains, were expressed in tryptophan auxotrophy RF12 strain (a gift from Robert Gennis & Toshio Iwasaki, Addgene plasmid # 62077) – .

Techniques:

Journal: Nature Communications

Article Title: Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

doi: 10.1038/s41467-023-44565-5

Figure Lengend Snippet: The 19 F solution NMR peaks of AppA, Oa PAC, and Sy PixD BLUF domains are colored red, blue and green, respectively. Their resolved peaks are marked with numbers (1, 2/2′, 3/3′), denoting three classified configurations. Peak 1 has been assigned to the W in NH in configuration, Peak 2 and 2′ have been assigned to subpopulations of the W in NH out configuration, and Peak 3 and 3′ have been assigned to subpopulations of the W out configuration. The chemical structure of 5FW with atomic numbering is depicted in the lower panel.

Article Snippet: The 5FW and 7FW labeled protein samples, i.e., WT and YnF mutants of Oa PAC 1-102 , Sy PixD 1-150 , and AppA 1-124 BLUF domains, were expressed in tryptophan auxotrophy RF12 strain (a gift from Robert Gennis & Toshio Iwasaki, Addgene plasmid # 62077) – .

Techniques:

Journal: Nature Communications

Article Title: Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

doi: 10.1038/s41467-023-44565-5

Figure Lengend Snippet: a – c Transient absorption 2D contour maps for AppA Y21F ( a ), Sy PixD Y8F ( b ), and Oa PAC Y6F ( c ) in H 2 O buffer. The upper panel shows the original TA 2D spectra and the lower panel shows the 2D spectra after subtracting the contribution from FMN * component. Detected transient species are denoted with dashed circles with internal annotations representing possible intermediates (FMN * , FMN˙ − /W˙, FMN˙ − /WH˙ + , FMNH˙/W˙). d – f Scaled transient absorption spectral (red and blue lines) snapshots at selected time delays after FMN * subtraction for AppA Y21F ( d ), Sy PixD Y8F ( e ), and Oa PAC Y6F ( f ) are shown to uncover the possible intermediates.

Article Snippet: The 5FW and 7FW labeled protein samples, i.e., WT and YnF mutants of Oa PAC 1-102 , Sy PixD 1-150 , and AppA 1-124 BLUF domains, were expressed in tryptophan auxotrophy RF12 strain (a gift from Robert Gennis & Toshio Iwasaki, Addgene plasmid # 62077) – .

Techniques:

Journal: Nature Communications

Article Title: Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

doi: 10.1038/s41467-023-44565-5

Figure Lengend Snippet: a – c columns show the target analysis results of AppA Y21F ( a ), Sy PixD Y8F ( b ), and Oa PAC Y6F ( c ) in H 2 O condition, respectively. The upper panels illustrate the proposed kinetic models, single rocking ( a ) and double rocking models ( b, c ), for the photoreaction cycle. The arrows depict pathways with fitted averaged lifetimes, while the symbol “×” in ( a ) indicates that PT2 is inaccessible within our time window for the AppA Y21F mutant. In the middle and lower panels, the species-associated differential spectra (SADS) and corresponding kinetic traces are presented; FMN * is depicted in red, FMN˙ − /WH˙ + in purple, FMN˙ − /W˙ in green, and FMNH˙/W˙ in blue. The inserts are the initial dynamics up to 50 ps.

Article Snippet: The 5FW and 7FW labeled protein samples, i.e., WT and YnF mutants of Oa PAC 1-102 , Sy PixD 1-150 , and AppA 1-124 BLUF domains, were expressed in tryptophan auxotrophy RF12 strain (a gift from Robert Gennis & Toshio Iwasaki, Addgene plasmid # 62077) – .

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

doi: 10.1038/s41467-023-44565-5

Figure Lengend Snippet: The 19 F NMR spectra of AppA, Oa PAC, and Sy PixD BLUF domains are shown in red, blue, and green, respectively. Their resolved peaks are marked with numbers (1/1′, 2/2′, 3), denoting three classified configurations. Peaks 1 and 1′ correspond to subpopulations of the W in NH in configuration, Peaks 2 and 2′ relate to subpopulations of the W in NH out configuration, while Peak 3 represents the W out configuration. The chemical structure of 7FW, along with atomic numbering, is illustrated in the lower panel.

Article Snippet: The 5FW and 7FW labeled protein samples, i.e., WT and YnF mutants of Oa PAC 1-102 , Sy PixD 1-150 , and AppA 1-124 BLUF domains, were expressed in tryptophan auxotrophy RF12 strain (a gift from Robert Gennis & Toshio Iwasaki, Addgene plasmid # 62077) – .

Techniques:

Journal: Nature Communications

Article Title: Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

doi: 10.1038/s41467-023-44565-5

Figure Lengend Snippet: a – c A stretched exponential function and a parallel kinetic model were adopted to analyze the kinetic traces at a selective probe wavelength of 760 nm for AppA W104F ( a ), Oa PAC W90F ( b ), and Sy PixD W91F ( c ) in H 2 O buffer. The raw data is presented as symbols (○), while the fitted traces are shown as straight lines. Decomposed dynamics of FMN * (red dashed lines) and 3 FMN (orange dashed lines) are shown up to 4000 ps with insets up to 250 ps.

Article Snippet: The 5FW and 7FW labeled protein samples, i.e., WT and YnF mutants of Oa PAC 1-102 , Sy PixD 1-150 , and AppA 1-124 BLUF domains, were expressed in tryptophan auxotrophy RF12 strain (a gift from Robert Gennis & Toshio Iwasaki, Addgene plasmid # 62077) – .

Techniques: