Automated Determination of Nuclear Magnetic Resonance Chemical Shift Perturbations in Ligand Screening Experiments: The PICASSO Web Server

Abstract

Nuclear magnetic resonance (NMR) is an effective, commonly used experimental approach to screen small organic molecules against a protein target. A very popular method consists of monitoring the changes of the NMR chemical shifts of the protein nuclei upon addition of the small molecule to the free protein. Multidimensional NMR experiments allow the interacting residues to be mapped along the protein sequence. A significant amount of human effort goes into manually tracking the chemical shift variations, especially when many signals exhibit chemical shift changes and when many ligands are tested. Some computational approaches to automate the procedure are available, but none of them as a web server. Furthermore, some methods require the adoption of a fairly specific experimental setup, such as recording a series of spectra at increasing small molecule:protein ratios. In this work, we developed a tool requesting a minimal amount of experimental data from the user, implemented it as an open-source program, and made it available as a web application. Our tool compares two spectra, one of the free protein and one of the small molecule:protein mixture, based on the corresponding peak lists. The performance of the tool in terms of correct identification of the protein-binding regions has been evaluated on different protein targets, using experimental data from interaction studies already available in the literature. For a total of 16 systems, our tool achieved between 79% and 100% correct assignments, properly identifying the protein regions involved in the interaction.

Site-Selective Functionalized PD-1 Mutant for a Modular Immunological Activity against Cancer Cells

“…This set of NMR data is consistent with a slow exchange regime on the NMR time scale previously observed and provides complementary information on the residues involved in the binding. To obtain the experimental restraints for the docking calculation of the adduct between HACTR-PD-1 and PD-L1, a prediction of the assignment of the resonances observed in the 2D 1 H– 15 N HSQC NMR spectra of the complex and the related chemical shift perturbation set (Figure A,B) were generated by using the PICASSO Web server . In the program, the 2D 1 H– 15 N HSQC reference spectra of free [U– 15 N] HACTR-PD-1 and free [U– 15 N] PD-L1 were compared to the spectra of [U– 15 N] HACTR-PD-1/PD-L1 and [U– 15 N] PD-L1/HACTR-PD-1 (in 1:1 molar ratio), respectively, to provide the assignment and the chemical shift perturbations on the two proteins in the complex.…”

“…To obtain the experimental restraints for the docking calculation of the adduct between HACTR-PD-1 and PD-L1, a prediction of the assignment of the resonances observed in the 2D 1 H− 15 N HSQC NMR spectra of the complex and the related chemical shift perturbation set (Figure 2A,B) were generated by using the PICASSO Web server. 26 In the program, the 2D 1 H− 15 N HSQC reference spectra of free [U− 15 N] HACTR-PD-1 and free [U− 15 N] PD-L1 were compared to the spectra of [U− 15 N] HACTR-PD-1/PD-L1 and [U− 15 N] PD-L1/ HACTR-PD-1 (in 1:1 molar ratio), respectively, to provide the assignment and the chemical shift perturbations on the two proteins in the complex. The residues experiencing the largest effects were imposed as "active residues" in the HADDOCK calculations (Figure 2C).…”

Fully Automated Characterization of Protein–Peptide Binding by Microfluidic 2D NMR

“…The current platform that is working with microliter volumes brings the analytical power of NMR in line with microliter protein protein expression systems , for generating fully automated discovery workflows. Further improvements could be realized by combining recent advances in autonomous processing of protein NMR data. − The modularity of the system and flexibility of the microfluidic chip design is such that the device can easily be adapted to suit specific experimental needs such as screening applications, protein–protein interactions and studies of multiligand equilibria. In addition, the device could be used for deuterium exchange experiments and protein-folding studies in response to buffer changes.…”

The Bifunctional Effects of Lactoferrin (LFcinB11) in Inhibiting Neural Cell Adhesive Molecule (NCAM) Polysialylation and the Release of Neutrophil Extracellular Traps (NETs)

“…where D N and D NH represent the changes in 15 N and 1 H chemical shifts, respectively, upon ligand binding [50][51][52][53][54][55][56][57][58].…”