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Unread 01-21-2016, 01:08 PM
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Default Combined ligand-observe (19)F and protein-observe (15)N,(1)H-HSQC NMR suggests phenylalanine as the key ?-somatostatin residue recognized by human protein disulfide isomerase.

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

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

Sci Rep. 2016;6:19518

Authors: Richards KL, Rowe ML, Hudson PB, Williamson RA, Howard MJ

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 ?-somatostatin (AGSKNFFWKTFTSS) binding to hPDI using (19)F ligand-observe and (15)N,(1)H-HSQC protein-observe NMR methods. Phe residues in ?-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 ?M 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 ?-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)N protein-observe but highlighted the advantage that (19)F offers improved Kd precision due to higher spectrum resolution and greater chemical environment sensitivity.


PMID: 26786784 [PubMed - in process]



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