¹⁹F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b′x from human protein disulphide isomerase (hPDI)

Abstract

Fluoroindole recombinant protein labelling enables a 19F NMR study to observe protein–ligand binding and dissociation constant determination.

Dual Labeling of the CBP/p300 KIX Domain for 19F NMR Leads to Identification of a New Small‐Molecule Binding Site

“…This dual-labeling strategy augments previously reported PrOF NMR methods for studying ligand-protein interactions [19] , small molecule screening [20] , as well as protein conformational changes [14, 21] and allostery [22] . Dual-labeling provides the ability to insert additional probes into the protein of interest and report on a new ligand binding mode.…”

“…In both cases, the 3FY resonances were very similar (0.04 ppm average difference) between the two spectra indicating a minimal change in chemical environment in the folded protein, despite the slightly lower molar ellipticity from CD. To date, this data is the first example of a dual-labeled protein analyzed by 19 F NMR. The single 4FF resonance found at −117.3 ppm is also in a similar region as previously reported in the singly-labeled protein.…”

“…[8b] In this study, fluorine labeling was high but still resulted in a heterogeneously labeled sample, which may have a significant effect on the resulting NMR spectrum. As such, a 19 F NMR study on a multi-labeled protein has yet to be reported. To address this need, we explored a dual-labeling strategy using both 3-fluorotyrosine (3FY) and 4-fluorophenylalanine (4FF) as a test case for characterizing native peptides sequences and newly discovered small molecules for the KIX domain for future chemical probe development.…”

Combined ligand-observe 19F and protein-observe 15N,1H-HSQC NMR suggests phenylalanine as the key Δ-somatostatin residue recognized by human protein disulfide isomerase

“…The only structural information on a PDI-ligand complex to date stems from the recently published thermophillic fungus ( Humicola insolens ) b’xa’ PDI bound to a α-synuclein peptide (αSN) that identified this PDI as being able to capture a hydrophobic ligand segment with key contacts from valine and leucine αSN residues 10 . However, this binding site identified in Humicola insolens b’ is located toward the N-terminal region of b’ whereas the binding site in hPDI b’ appears much larger and registers across the beta-sheet and residues in the C-terminal half of the protein including the x- linker 5 6 15 .…”

“…The overlaid spectra inform on the binding event and highlight the residues involved. 19 F NMR has re-emerged as an important and valuable method for studying proteins and biomolecules as illustrated by several recent publications 15 18 19 20 21 22 23 24 25 . We report the combined use of backbone 15 N/ 1 H and 19 F NMR chemical shift perturbations to study the molecular detail and specificity of the ligand binding mechanism of the hPDI fragment b’x with a known peptide ligand, Δ-somatostatin (Δ-som; AGSKNFFWKTFTSS) 2 26 .…”