Journal: Genes

Article Title: Structural Insight into the Mechanism of PALB2 Interaction with MRG15

doi: 10.3390/genes12122002

Figure Lengend Snippet: MRG interaction with PALB2. ( A ) Preliminary analysis of MRG binding by purified His-tagged PALB2 peptides represented schematically as black lines with numbers for N- and C-terminal amino acids shown at the ends and with the FQLP motif highlighted in red. Panel on top shows predicted secondary structure elements (α-helixes as cylinders and β-strands as arrows). Panel on the bottom shows secondary structure elements observed in a crystal structure. ( B ) Anisotropy isotherm of FAM-PALB2 597−630 peptide titration of MRG.

Article Snippet: K d was calculated with GraphPad Prism software by fitting the data with a non-linear regression analysis using a standard four-parameter logistic equation to identify K d . (1) y = y m i n + y m a x – y m i n 1 + 10 ( l o g E C 50 − X ) x n where y min and y max are the minimum and maximum anisotropy values, X represents the log concentration of protein, n represents the Hill slope, and EC50 is equal to K d . R 2 is determined by the Prism software by computing the sum of the squares of the distances of the points from the best-fit curve determined by a nonlinear regression model. For competition assays, mutant PALB2 597−630 peptides (Genscript Biotech, Piscataway, NJ, USA) were dissolved in DMSO and serially diluted from 10 μM to 0.6 nM in 20 μL of 25 mM HEPES pH 8.0, 1 mM TCEP, 10% DMSO, and 200 mM NaCl.

Techniques: Binding Assay, Purification, Titration

Journal: Genes

Article Title: Structural Insight into the Mechanism of PALB2 Interaction with MRG15

doi: 10.3390/genes12122002

Figure Lengend Snippet: Crystal structure of MRG PALB2 complex. ( A ) Cartoon representation of two complexes in an asymmetric unit, with MRG chains shown in green and magenta and PALB2 peptides in cyan and yellow. N- and C-terminal amino acids modeled for each PALB2 peptide are shown. ( B ) Two complexes were superimposed by the MRG subunits (shown in the grey surface representation), resulting in the overlap of two PALB2 597−630 peptides.

Article Snippet: K d was calculated with GraphPad Prism software by fitting the data with a non-linear regression analysis using a standard four-parameter logistic equation to identify K d . (1) y = y m i n + y m a x – y m i n 1 + 10 ( l o g E C 50 − X ) x n where y min and y max are the minimum and maximum anisotropy values, X represents the log concentration of protein, n represents the Hill slope, and EC50 is equal to K d . R 2 is determined by the Prism software by computing the sum of the squares of the distances of the points from the best-fit curve determined by a nonlinear regression model. For competition assays, mutant PALB2 597−630 peptides (Genscript Biotech, Piscataway, NJ, USA) were dissolved in DMSO and serially diluted from 10 μM to 0.6 nM in 20 μL of 25 mM HEPES pH 8.0, 1 mM TCEP, 10% DMSO, and 200 mM NaCl.

Techniques:

Journal: Genes

Article Title: Structural Insight into the Mechanism of PALB2 Interaction with MRG15

doi: 10.3390/genes12122002

Figure Lengend Snippet: Hydrophobic interactions of the core signature motif. ( A ) Cartoon representation of PALB2 peptide in yellow with the conserved hydrophobic amino acids of the FxLP motif interaction with MRG shown in the surface representation and color-coded according to surface electrostatic potential. ( B ) Conformation comparisons of three hydrophobic amino acids of the FxLPxxФ motif from three different MRG-interacting peptides with carbon atoms of PALB2 shown in yellow, of 2N1D shown in green, and of 2INE in orange. All complexes were superimposed by MRG subunits shown as in panel A.

Article Snippet: K d was calculated with GraphPad Prism software by fitting the data with a non-linear regression analysis using a standard four-parameter logistic equation to identify K d . (1) y = y m i n + y m a x – y m i n 1 + 10 ( l o g E C 50 − X ) x n where y min and y max are the minimum and maximum anisotropy values, X represents the log concentration of protein, n represents the Hill slope, and EC50 is equal to K d . R 2 is determined by the Prism software by computing the sum of the squares of the distances of the points from the best-fit curve determined by a nonlinear regression model. For competition assays, mutant PALB2 597−630 peptides (Genscript Biotech, Piscataway, NJ, USA) were dissolved in DMSO and serially diluted from 10 μM to 0.6 nM in 20 μL of 25 mM HEPES pH 8.0, 1 mM TCEP, 10% DMSO, and 200 mM NaCl.

Techniques:

Journal: Genes

Article Title: Structural Insight into the Mechanism of PALB2 Interaction with MRG15

doi: 10.3390/genes12122002

Figure Lengend Snippet: Contribution of conserved amino acids and cancer-associated inherited mutations to the PALB2 interaction with MRG15. ( A , B ) Two orthogonal views of the PALB2 interaction with MRG15, highlighting the residues identified in cancer patients shown in cyan and the binding hydrophobic core shown in orange. ( C ) Displacement of WT FAM-PALB2 peptide at 10 nM concentration from MRG with titration by mutant PALB2 peptides. ( D ) Dissociation constants Ki for each mutant calculated from titration isotherms shown in ( C ).

Article Snippet: K d was calculated with GraphPad Prism software by fitting the data with a non-linear regression analysis using a standard four-parameter logistic equation to identify K d . (1) y = y m i n + y m a x – y m i n 1 + 10 ( l o g E C 50 − X ) x n where y min and y max are the minimum and maximum anisotropy values, X represents the log concentration of protein, n represents the Hill slope, and EC50 is equal to K d . R 2 is determined by the Prism software by computing the sum of the squares of the distances of the points from the best-fit curve determined by a nonlinear regression model. For competition assays, mutant PALB2 597−630 peptides (Genscript Biotech, Piscataway, NJ, USA) were dissolved in DMSO and serially diluted from 10 μM to 0.6 nM in 20 μL of 25 mM HEPES pH 8.0, 1 mM TCEP, 10% DMSO, and 200 mM NaCl.

Techniques: Binding Assay, Concentration Assay, Titration, Mutagenesis

Journal: Genes

Article Title: Structural Insight into the Mechanism of PALB2 Interaction with MRG15

doi: 10.3390/genes12122002

Figure Lengend Snippet: Comparison of PALB2 peptide conformation with other MRG-binding peptides. Two orthogonal views of MRG (shown in grey surface representation) complexes with PALB2 (yellow), MSL1 (magenta), MRGBP (green), ASH1L (orange) and Pf1 (cyan) shown in cartoon representations with the middle part of the peptides shown in panel ( A ) and the N-terminal parts in panel ( B ). All structures were superimposed by MRG domain.

Article Snippet: K d was calculated with GraphPad Prism software by fitting the data with a non-linear regression analysis using a standard four-parameter logistic equation to identify K d . (1) y = y m i n + y m a x – y m i n 1 + 10 ( l o g E C 50 − X ) x n where y min and y max are the minimum and maximum anisotropy values, X represents the log concentration of protein, n represents the Hill slope, and EC50 is equal to K d . R 2 is determined by the Prism software by computing the sum of the squares of the distances of the points from the best-fit curve determined by a nonlinear regression model. For competition assays, mutant PALB2 597−630 peptides (Genscript Biotech, Piscataway, NJ, USA) were dissolved in DMSO and serially diluted from 10 μM to 0.6 nM in 20 μL of 25 mM HEPES pH 8.0, 1 mM TCEP, 10% DMSO, and 200 mM NaCl.

Techniques: Comparison, Binding Assay