Journal: Journal of the American Chemical Society

Article Title: Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein–Ligand Interactions from a Single NMR Sample

doi: 10.1021/jacs.3c02218

Figure Lengend Snippet: Sketch of the Imaging STD NMR approach for the determination of dissociation constants based on initial growth of the build-up curves. An STD-NMR build-up curve is extracted at each depth of the tube, corresponding to increasing [ligand]/[protein] ratios (bottom to top). From these, the initial slopes are calculated and plotted against the ligand concentration to obtain the binding isotherm and hence K D .

Article Snippet: Experimental section; mathematical derivation of STD-AF 0 and of K D from the STD-AF 0 -binding isotherm; imaging STD NMR control experiments and comparison of STD NMR and imaging NMR spectra; build-up curves with data and binding isotherms data for the three complexes; mathematical derivation of more general fitting for K D determination; K D values at single saturation times; comparison for instrument time for STD NMR titration and imaging STD NMR; binding epitope mappings at increasing concentrations for W/BSA and GlcNAc/WGA; pulse sequence for the STD CSI experiment (Bruker); amount of mass and concentration for gradient formation of small molecules; effect of DMSO in the stock of the diffusion profile; effect of the protein concentration on sensitivity and simulation of protein diffusion; macros for automated imaging STD NMR data processing (Bruker); STD NMR data processing: manual method and automation; and STD NMR data processing on Mnova 14.3.1 by line fitting ( PDF ) Calculation of mass and concentration of ligands and for imaging STD NMR data processing ( XLSX )

Techniques: Imaging, Concentration Assay, Binding Assay

Journal: Journal of the American Chemical Society

Article Title: Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein–Ligand Interactions from a Single NMR Sample

doi: 10.1021/jacs.3c02218

Figure Lengend Snippet: (a) Example concentration gradient produced by placing 50 μL of methyl orange solution on top of 400 μL of BSA protein in buffer. (b) Diffusion profile of tryptophan between 22 and 26 h, showing the stability of the concentration gradient needed for analysis by imaging STD NMR. Depth is the distance from the boundary, where the tryptophan solution was layered (31 mm from the tube bottom).

Article Snippet: Experimental section; mathematical derivation of STD-AF 0 and of K D from the STD-AF 0 -binding isotherm; imaging STD NMR control experiments and comparison of STD NMR and imaging NMR spectra; build-up curves with data and binding isotherms data for the three complexes; mathematical derivation of more general fitting for K D determination; K D values at single saturation times; comparison for instrument time for STD NMR titration and imaging STD NMR; binding epitope mappings at increasing concentrations for W/BSA and GlcNAc/WGA; pulse sequence for the STD CSI experiment (Bruker); amount of mass and concentration for gradient formation of small molecules; effect of DMSO in the stock of the diffusion profile; effect of the protein concentration on sensitivity and simulation of protein diffusion; macros for automated imaging STD NMR data processing (Bruker); STD NMR data processing: manual method and automation; and STD NMR data processing on Mnova 14.3.1 by line fitting ( PDF ) Calculation of mass and concentration of ligands and for imaging STD NMR data processing ( XLSX )

Techniques: Concentration Assay, Produced, Diffusion-based Assay, Imaging

Journal: Journal of the American Chemical Society

Article Title: Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein–Ligand Interactions from a Single NMR Sample

doi: 10.1021/jacs.3c02218

Figure Lengend Snippet: Spectra from imaging STD NMR experiments of a sample containing a gradient of tryptophan against homogeneous concentrations of BSA. (a) 1D NMR spectrum of the sample acquired with a 30° pulse, without water suppression. (b) On- and off-resonance spectra and (c) STD difference spectra of slice 8 of 16 of the imaging STD experiment performed on the same sample. The imaging STD NMR experiment was acquired with eight scans for an experimental time of 22 min.

Article Snippet: Experimental section; mathematical derivation of STD-AF 0 and of K D from the STD-AF 0 -binding isotherm; imaging STD NMR control experiments and comparison of STD NMR and imaging NMR spectra; build-up curves with data and binding isotherms data for the three complexes; mathematical derivation of more general fitting for K D determination; K D values at single saturation times; comparison for instrument time for STD NMR titration and imaging STD NMR; binding epitope mappings at increasing concentrations for W/BSA and GlcNAc/WGA; pulse sequence for the STD CSI experiment (Bruker); amount of mass and concentration for gradient formation of small molecules; effect of DMSO in the stock of the diffusion profile; effect of the protein concentration on sensitivity and simulation of protein diffusion; macros for automated imaging STD NMR data processing (Bruker); STD NMR data processing: manual method and automation; and STD NMR data processing on Mnova 14.3.1 by line fitting ( PDF ) Calculation of mass and concentration of ligands and for imaging STD NMR data processing ( XLSX )

Techniques: Imaging

Journal: Journal of the American Chemical Society

Article Title: Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein–Ligand Interactions from a Single NMR Sample

doi: 10.1021/jacs.3c02218

Figure Lengend Snippet: Imaging STD NMR Langmuir binding isotherms for K D determination of the (a) tryptophan/BSA complex, based on the average of all the aromatic protons; (b) and GlcNAc/WGA, based on the methyl group signal; and (c) 3NPG/CTB complex, based on the H2,3,5 proton signal of the sugar ring. For the three complexes, we show the binding isotherms (lines) obtained from fitting either the initial slopes of build-up curves (STD-AF0), in blue dots, or from the STD-AF at increasing saturation time, in orange to brown dots. Tabulated data for the binding isotherms are reported in Section S4 of the Supporting Information , where the STD NMR build-up curves obtained at each depth of the tube, i.e., at increasing ligand concentration, and tabulated data, are also included.

Article Snippet: Experimental section; mathematical derivation of STD-AF 0 and of K D from the STD-AF 0 -binding isotherm; imaging STD NMR control experiments and comparison of STD NMR and imaging NMR spectra; build-up curves with data and binding isotherms data for the three complexes; mathematical derivation of more general fitting for K D determination; K D values at single saturation times; comparison for instrument time for STD NMR titration and imaging STD NMR; binding epitope mappings at increasing concentrations for W/BSA and GlcNAc/WGA; pulse sequence for the STD CSI experiment (Bruker); amount of mass and concentration for gradient formation of small molecules; effect of DMSO in the stock of the diffusion profile; effect of the protein concentration on sensitivity and simulation of protein diffusion; macros for automated imaging STD NMR data processing (Bruker); STD NMR data processing: manual method and automation; and STD NMR data processing on Mnova 14.3.1 by line fitting ( PDF ) Calculation of mass and concentration of ligands and for imaging STD NMR data processing ( XLSX )

Techniques: Imaging, Binding Assay, Concentration Assay

Journal: Journal of the American Chemical Society

Article Title: Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein–Ligand Interactions from a Single NMR Sample

doi: 10.1021/jacs.3c02218

Figure Lengend Snippet: Assessment of binding specificity by imaging STD NMR. Top: cartoons of how STD NMR binding epitopes can be used for assessing the specificity of binding, where a specific protein–ligand complex is represented in (a), and a non-specific protein–ligand complex is represented in (b). Bottom: histograms of the binding epitope mapping of the complexes GlcNAc/WGA (top) and W/BSA (bottom) obtained from initial slopes derived from imaging STD NMR build-up curves at increasing ligand concentration, from a single tube. For the atom nomenclature, see Figure . GlcNAc-binding epitopes are normalized to the methyl group which gave the strongest STD response. The strongest STD response exhibited by the tryptophan changed for each concentration due to non-specific binding. Tabulated data are reported in Section S8 .

Article Snippet: Experimental section; mathematical derivation of STD-AF 0 and of K D from the STD-AF 0 -binding isotherm; imaging STD NMR control experiments and comparison of STD NMR and imaging NMR spectra; build-up curves with data and binding isotherms data for the three complexes; mathematical derivation of more general fitting for K D determination; K D values at single saturation times; comparison for instrument time for STD NMR titration and imaging STD NMR; binding epitope mappings at increasing concentrations for W/BSA and GlcNAc/WGA; pulse sequence for the STD CSI experiment (Bruker); amount of mass and concentration for gradient formation of small molecules; effect of DMSO in the stock of the diffusion profile; effect of the protein concentration on sensitivity and simulation of protein diffusion; macros for automated imaging STD NMR data processing (Bruker); STD NMR data processing: manual method and automation; and STD NMR data processing on Mnova 14.3.1 by line fitting ( PDF ) Calculation of mass and concentration of ligands and for imaging STD NMR data processing ( XLSX )

Techniques: Binding Assay, Imaging, Derivative Assay, Concentration Assay