Study of hydrophobic interactions between acylated proteins and phospholipid bilayers using BIACORE

Formation of a High Affinity Lipid-Binding Intermediate during the Early Aggregation Phase of α-Synuclein

“…Because of the fast on rates and long off rates, direct fitting of the sensorgrams was not practical. Scatchard analysis of WT and mutant α-syn were nonlinear, indicating a two-state system and this was interpreted as multiple high- and low-affinity binding interactions ( , ). On this basis the following model was used to describe the observed data…”

Formation of a High Affinity Lipid-Binding Intermediate during the Early Aggregation Phase of α-Synuclein

“…Because of the fast on rates and long off rates, direct fitting of the sensorgrams was not practical. Scatchard analysis of WT and mutant α-syn were nonlinear, indicating a two-state system and this was interpreted as multiple high- and low-affinity binding interactions ( , ). On this basis the following model was used to describe the observed data…”

Differential Mechanisms for Calcium-Dependent Protein/Membrane Association as Evidenced from SPR-Binding Studies on Supported Biomimetic Membranes

“…A monolayer of 1-octadecane thiol was self-assembled on top of the gold surface to facilitate subsequent vesicle fusion via hydrophobic interactions (12,29,53). In previous experiments of 1-octadecane thiol adsorption onto gold surfaces, layer thicknesses of 29 ( 2.5 Å have been determined (12,54,55). In our experimental design, best SPR curve fits with the Winspall program were obtained for an 1-octadecane thiol inner monolayer thickness of 29 ( 2.5 Å and a refractive index of 1.5.…”

“…Hemimembrane formation was obtained by injection the egg-PC vesicle suspension (4 mL at a flow rate of about 3 mL min -1 ) over the surface. The cell was first washed with NaOH (4 mM, 5 mL) which was shown to stabilize the bilayer (29,53,54), and finally the cell was flushed during 1 h with 20 mM HEPES-Na, pH 7.5, 150 mM NaCl, at a flow rate of 1 mL min -1 .…”

Comparative Studies on Bulky DNA Damage Binding by Nucleotide Excision Repair Proteins Using Surface Plasmon Resonance, Differential Scanning Fluorometry, and DNase I Footprinting

“…The SPR experiments were triplicated, with each duplex injected twice at the indicated 1:1 Langmuir model by the BIAevaluation software package V3.2, provided by the manufacturer. 28,29 Processing involved data zeroing, cropping, aligning injection times, binding curve fitting, and off-rate analysis. The binding affinity (K D ), association rate (k a ), and dissociation rate (k d ) averages, along with their respective standard deviations, were calculated using the steadystate affinity analysis.…”

Formation of a High Affinity Lipid-Binding Intermediate during the Early Aggregation Phase of α-Synuclein

“…Because of the fast on rates and long off rates, direct fitting of the sensorgrams was not practical. Scatchard analysis of WT and mutant α-syn were nonlinear, indicating a two-state system and this was interpreted as multiple high- and low-affinity binding interactions ( , ). On this basis the following model was used to describe the observed data…”

Differential Mechanisms for Calcium-Dependent Protein/Membrane Association as Evidenced from SPR-Binding Studies on Supported Biomimetic Membranes

“…A monolayer of 1-octadecane thiol was self-assembled on top of the gold surface to facilitate subsequent vesicle fusion via hydrophobic interactions (12,29,53). In previous experiments of 1-octadecane thiol adsorption onto gold surfaces, layer thicknesses of 29 ( 2.5 Å have been determined (12,54,55). In our experimental design, best SPR curve fits with the Winspall program were obtained for an 1-octadecane thiol inner monolayer thickness of 29 ( 2.5 Å and a refractive index of 1.5.…”

“…Hemimembrane formation was obtained by injection the egg-PC vesicle suspension (4 mL at a flow rate of about 3 mL min -1 ) over the surface. The cell was first washed with NaOH (4 mM, 5 mL) which was shown to stabilize the bilayer (29,53,54), and finally the cell was flushed during 1 h with 20 mM HEPES-Na, pH 7.5, 150 mM NaCl, at a flow rate of 1 mL min -1 .…”

Comparative Studies on Bulky DNA Damage Binding by Nucleotide Excision Repair Proteins Using Surface Plasmon Resonance, Differential Scanning Fluorometry, and DNase I Footprinting

“…The SPR experiments were triplicated, with each duplex injected twice at the indicated 1:1 Langmuir model by the BIAevaluation software package V3.2, provided by the manufacturer. 28,29 Processing involved data zeroing, cropping, aligning injection times, binding curve fitting, and off-rate analysis. The binding affinity (K D ), association rate (k a ), and dissociation rate (k d ) averages, along with their respective standard deviations, were calculated using the steadystate affinity analysis.…”

Formation of a High Affinity Lipid-Binding Intermediate during the Early Aggregation Phase of α-Synuclein

“…Because of the fast on rates and long off rates, direct fitting of the sensorgrams was not practical. Scatchard analysis of WT and mutant α-syn were nonlinear, indicating a two-state system and this was interpreted as multiple high- and low-affinity binding interactions ( , ). On this basis the following model was used to describe the observed data…”

Differential Mechanisms for Calcium-Dependent Protein/Membrane Association as Evidenced from SPR-Binding Studies on Supported Biomimetic Membranes

“…A monolayer of 1-octadecane thiol was self-assembled on top of the gold surface to facilitate subsequent vesicle fusion via hydrophobic interactions (12,29,53). In previous experiments of 1-octadecane thiol adsorption onto gold surfaces, layer thicknesses of 29 ( 2.5 Å have been determined (12,54,55). In our experimental design, best SPR curve fits with the Winspall program were obtained for an 1-octadecane thiol inner monolayer thickness of 29 ( 2.5 Å and a refractive index of 1.5.…”

“…Hemimembrane formation was obtained by injection the egg-PC vesicle suspension (4 mL at a flow rate of about 3 mL min -1 ) over the surface. The cell was first washed with NaOH (4 mM, 5 mL) which was shown to stabilize the bilayer (29,53,54), and finally the cell was flushed during 1 h with 20 mM HEPES-Na, pH 7.5, 150 mM NaCl, at a flow rate of 1 mL min -1 .…”

Comparative Studies on Bulky DNA Damage Binding by Nucleotide Excision Repair Proteins Using Surface Plasmon Resonance, Differential Scanning Fluorometry, and DNase I Footprinting

“…The SPR experiments were triplicated, with each duplex injected twice at the indicated 1:1 Langmuir model by the BIAevaluation software package V3.2, provided by the manufacturer. 28,29 Processing involved data zeroing, cropping, aligning injection times, binding curve fitting, and off-rate analysis. The binding affinity (K D ), association rate (k a ), and dissociation rate (k d ) averages, along with their respective standard deviations, were calculated using the steadystate affinity analysis.…”