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

Scientific Reports
Kirsty L RichardsMark J Howard

Abstract

Human protein disulphide isomerase (hPDI) is an endoplasmic reticulum (ER) based isomerase and folding chaperone. Molecular detail of ligand recognition and specificity of hPDI are poorly understood despite the importance of the hPDI for folding secreted proteins and its implication in diseases including cancer and lateral sclerosis. We report a detailed study of specificity, interaction and dissociation constants (Kd) of the peptide-ligand Δ-somatostatin (AGSKNFFWKTFTSS) binding to hPDI using (19)F ligand-observe and (15)N,(1)H-HSQC protein-observe NMR methods. Phe residues in Δ-somatostatin are hypothesised as important for recognition by hPDI therefore, step-wise peptide Phe-to-Ala changes were progressively introduced and shown to raise the Kd from 103 + 47 μM until the point where binding was abolished when all Phe residues were modified to Ala. The largest step-changes in Kd involved the F11A peptide modification which implies the C-terminus of Δ-somatostatin is a prime recognition region. Furthermore, this study also validated the combined use of (19)F ligand-observe and complimentary (15)N,(1)H-HSQC titrations to monitor interactions from the protein's perspective. (19)F ligand-observe NMR was ratified as mirroring (15)...Continue Reading

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Citations

Sep 15, 2016·ACS Chemical Biology·Andrew K UrickWilliam C K Pomerantz
Dec 16, 2016·Protein Science : a Publication of the Protein Society·Rachel D Cohen, Gary J Pielak
Jul 22, 2016·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Raymond S NortonChristopher A MacRaild
Dec 4, 2020·Protein Science : a Publication of the Protein Society·Gabriel J Fuente-GómezElizabeth E Howell
Feb 11, 2021·Beilstein Journal of Organic Chemistry·Diana GimenezSteven L Cobb
Oct 3, 2018·Biochemistry·Sanne J M VerhoorkChristopher R Coxon

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Methods Mentioned

BETA
protein folding
NMR
protein-folding
interaction studies

Software Mentioned

Bruker Topspin
NMRPipe
Synergy
KaleidaGraph
CCPN Analysis

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