Journal: International Journal of Molecular Sciences

Article Title: Association of Alpha-Crystallin with Human Cortical and Nuclear Lens Lipid Membrane Increases with the Grade of Cortical and Nuclear Cataract

doi: 10.3390/ijms25031936

Figure Lengend Snippet: ( A ) EPR spectra, taken at 37 °C, of 4PT in 64 yo male right eye lens CMs in the absence of α-crystallin (black) and with 23.036 μM α-crystallin (red). The amount of cortical total lipids (lipid plus Chol) used in CM sample was 0.15 mg. The ratio of the peak-to-peak intensity of the high-field line (h − ) and the central line (h 0 ) provides the mobility parameter (h − /h 0 ). The horizontal distance between the low- and high-field lines provides the maximum splitting. ( B ) Magnified image of the high-field line of the EPR spectra shown in ( A ), representing the unbound (U 0 ) and unbound plus bound (U 0 + B 0 ) contributions. The change in the peak-to-peak intensity of the high-field line of the EPR spectra was used to calculate the percentage of the membrane surface occupied (MSO) by α-crystallin. ( C ) EPR spectra, taken at −165 °C, of 4PT in a 73 yo male left eye lens CM in the absence of α-crystallin (black) and with 23.036 μM α-crystallin (red), showing the horizontal distance between the low-field and high-field lines, being used to measure 2A Z , which is a measure for hydrophobicity.

Article Snippet: 4-palmitamido-TEMPO (4PT) spin-label was acquired dissolved in chloroform from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Membrane

Journal: bioRxiv

Article Title: Fundamental steps in Minichromosome Maintenance complex assembly are conserved from archaea to eukaryotes

doi: 10.1101/2023.08.11.552962

Figure Lengend Snippet: a Overview of the FRET-based biochemical activity screen . Protein is pre-equilibrated with a forked DNA substrate. Addition of ATP/Mg 2+ initiates substrate unwinding, which spatially separates a fluorophore (Cy3) and quencher (BHQ2) causing an increase in fluorescence (λ = 570 nm). A scavenger strand (blue) prevents reannealing. b Percentage DNA unwound by each MCM sample at 25 °C (black) and 45 °C (grey) after 30 minutes. Unwinding was quantified by subtracting a no helicase control and then standardizing against a maximum fluorescence well, containing non-annealed Cy3-labelled ssDNA. Bars represent mean unwinding (n = 4). Error bars correspond to ± 1 sem.

Article Snippet: Fluorescein-labelled ssDNA substrate (5’[FAM]-pT 50 ; Merck) was used in all SEC experiments to allow us to characterise complexes formed on DNA in the absence of any unwinding.

Techniques: Activity Assay, Fluorescence

Journal: bioRxiv

Article Title: Fundamental steps in Minichromosome Maintenance complex assembly are conserved from archaea to eukaryotes

doi: 10.1101/2023.08.11.552962

Figure Lengend Snippet: The loading of Mac MCM constructs on to DNA was analysed by analytical SEC. Protein samples (10 µM) were pre-incubated with or without an equimolar ratio of fluorescein labelled ssDNA substrate (polyT 50 ) before application to a Superose 6 Increase 10/300 GL SEC column. Where stated, ATP at 1 mM and Mg 2+ at 10 mM were added to the buffer. UV absorbance was monitored at both 290 nm (solid trace) and 495 nm (dotted trace). Vertical dotted lines indicate the expected elution volumes of MCM oligomers with 1 to 6 subunits. a Mac MCM, b Mac MCM ΔWHD , c Mac MCM E391Q and d Mac MCM E391Q.ΔWHD

Article Snippet: Fluorescein-labelled ssDNA substrate (5’[FAM]-pT 50 ; Merck) was used in all SEC experiments to allow us to characterise complexes formed on DNA in the absence of any unwinding.

Techniques: Construct, Incubation

Journal: Current Research in Structural Biology

Article Title: Fragment-based exploration of the 14-3-3/Amot-p130 interface

doi: 10.1016/j.crstbi.2021.12.003

Figure Lengend Snippet: Comparison of hit fragment 22 activity over different 14-3-3 binding epitopes. (A) Overview of the selected 14-3-3 partner and their binding epitopes observed in the crystal structures. (B) Overlay of the crystal structures of the 14-3-3 binding partners (cartoon representation). The phosphorylated site and C-terminus of Amot-p130 and Ataxin are highlighted with stick representations. Fragment 22 is depicted with purple lines and surface. 14-3-3σΔC protein is always depicted as a white surface. (C – E) Crystal structures of fragment 22 (purple sticks and surface) bound to 14-3-3σΔC in complex with Amot-p130 peptide (cyan sticks and surface) PDB ID: 7NNE or in complex with p53 peptide (green sticks and surface) PDB ID: 6RM7 ( Guillory et al., 2020 ). (D) Binding mode of the Ataxin peptide (orange sticks and surface) PDB ID: 6QIU ( Leysen et al., 2021 ). The polar contacts between Arg781 of the peptide and Glu14 of 14-3-3 are depicted as black dashed lines. Glu14 of 14-3-3 is highlighted with red sticks and surface. (F) FP data (mean; triplicates) of 14-3-3η titration in presence and absence of 1 ​mM 22 and TAMRA-labelled Amot-p130, Ataxin, or p53 peptides. Background polarization was subtracted from all values. DMSO in the assay was 1%. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The 32-mer TAMRA labelled p53 peptide [ 362 SRAHSSHLKSKKGQSTSRHKKLMFK{pT}EGPDSD 393 ] was purchased from AnaSpec.

Techniques: Comparison, Activity Assay, Binding Assay, Titration