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

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


          We report a protein-observe 19F NMR-based ligand titration binding study of human PDI b′x with Δ-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single 19F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 μM.

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          Most cited references 21

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            Using chemical shift perturbation to characterise ligand binding.

            Chemical shift perturbation (CSP, chemical shift mapping or complexation-induced changes in chemical shift, CIS) follows changes in the chemical shifts of a protein when a ligand is added, and uses these to determine the location of the binding site, the affinity of the ligand, and/or possibly the structure of the complex. A key factor in determining the appearance of spectra during a titration is the exchange rate between free and bound, or more specifically the off-rate koff. When koff is greater than the chemical shift difference between free and bound, which typically equates to an affinity Kd weaker than about 3μM, then exchange is fast on the chemical shift timescale. Under these circumstances, the observed shift is the population-weighted average of free and bound, which allows Kd to be determined from measurement of peak positions, provided the measurements are made appropriately. (1)H shifts are influenced to a large extent by through-space interactions, whereas (13)Cα and (13)Cβ shifts are influenced more by through-bond effects. (15)N and (13)C' shifts are influenced both by through-bond and by through-space (hydrogen bonding) interactions. For determining the location of a bound ligand on the basis of shift change, the most appropriate method is therefore usually to measure (15)N HSQC spectra, calculate the geometrical distance moved by the peak, weighting (15)N shifts by a factor of about 0.14 compared to (1)H shifts, and select those residues for which the weighted shift change is larger than the standard deviation of the shift for all residues. Other methods are discussed, in particular the measurement of (13)CH3 signals. Slow to intermediate exchange rates lead to line broadening, and make Kd values very difficult to obtain. There is no good way to distinguish changes in chemical shift due to direct binding of the ligand from changes in chemical shift due to allosteric change. Ligand binding at multiple sites can often be characterised, by simultaneous fitting of many measured shift changes, or more simply by adding substoichiometric amounts of ligand. The chemical shift changes can be used as restraints for docking ligand onto protein. By use of quantitative calculations of ligand-induced chemical shift changes, it is becoming possible to determine not just the position but also the orientation of ligands. Copyright © 2013 Elsevier B.V. All rights reserved.
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              Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation.

              Disulfide bond formation is probably involved in the biogenesis of approximately one third of human proteins. A central player in this essential process is protein disulfide isomerase or PDI. PDI was the first protein-folding catalyst reported. However, despite more than four decades of study, we still do not understand much about its physiological mechanisms of action. This review examines the published literature with a critical eye. This review aims to (a) provide background on the chemistry of disulfide bond formation and rearrangement, including the concept of reduction potential, before examining the structure of PDI; (b) detail the thiol-disulfide exchange reactions that are catalyzed by PDI in vitro, including a critical examination of the assays used to determine them; (c) examine oxidation and reduction of PDI in vivo, including not only the role of ERo1 but also an extensive assessment of the role of glutathione, as well as other systems, such as peroxide, dehydroascorbate, and a discussion of vitamin K-based systems; (d) consider the in vivo reactions of PDI and the determination and implications of the redox state of PDI in vivo; and (e) discuss other human and yeast PDI-family members.

                Author and article information

                Org Biomol Chem
                Org. Biomol. Chem
                Organic & Biomolecular Chemistry
                Royal Society of Chemistry
                21 June 2014
                6 May 2014
                : 12
                : 23
                : 3808-3812
                [a ] Protein Science Group , School of Biosciences , University of Kent , Giles Lane , Canterbury , Kent CT2 7NJ , UK . Email: r.a.williamson@ ; Email: m.j.howard@
                This journal is © The Royal Society of Chemistry 2014

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.



                †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ob00699b

                Organic & Biomolecular chemistry


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