Dataset Information

Probing Peptidylprolyl Bond cis/trans Status Using Distal ¹⁹ F NMR Reporters.

ABSTRACT: A method for measuring peptidylprolyl bond cis-trans conformational status in peptide models is described, using 4-fluorophenylalanine (4FPhe) as a distal reporter for ¹⁹ F NMR. The %cis-Pro population was measured for peptides of the general structure Ac-X-Pro-Z-Ala-Ala-4FPhe (X and Z are proteinogenic amino acids) at pH 7.4, and provided conformational populations consistent with literature values obtained by more complex methods. This approach was applied to probe the prolyl bond status in pentapeptide models of the intrinsically disordered C-terminal region of α-synuclein, which mirrored the preferences in the Ac-X-Pro-Z-Ala-4FPhe models. Advantageously, the ¹⁹ F reporter group does not need to be adjacent to or attached to proline to provide quantifiable signals and distal 4-fluorophenylalanines can be placed so as not to influence prolyl bond conformation. Finally, we demonstrated that the prolyl bond status is not significantly affected by pH when there are ionisable amino acid residues at the carboxyl side of proline, which makes ¹⁹ F NMR an invaluable tool with which to study proline isomerism at a range of pHs and in different solvents and buffers.

SUBMITTER: Killoran PM

PROVIDER: S-EPMC10946801 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Probing Peptidylprolyl Bond cis/trans Status Using Distal <sup>19</sup> F NMR Reporters.

Killoran Patrick M PM Hanson George S M GSM Verhoork Sanne J M SJM Smith Madeleine M Del Gobbo Davide D Lian Lu-Yun LY Coxon Christopher R CR

Chemistry (Weinheim an der Bergstrasse, Germany) 20230216 16

A method for measuring peptidylprolyl bond cis-trans conformational status in peptide models is described, using 4-fluorophenylalanine (4FPhe) as a distal reporter for <sup>19</sup> F NMR. The %cis-Pro population was measured for peptides of the general structure Ac-X-Pro-Z-Ala-Ala-4FPhe (X and Z are proteinogenic amino acids) at pH 7.4, and provided conformational populations consistent with literature values obtained by more complex methods. This approach was applied to probe the prolyl bond s ...[more]

PMID: 36550088

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Project description:Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate constants of the slow phase were determined. We conclude that the cis to trans interconversion of the pipecolinyl bond of the three peptidomacrolides may be responsible for the slow process. There was no indication of a suicide catalysis of this process by FKBPs.

Project description:The recently discovered macrolide FK506 has been demonstrated to have potent immunosuppressive activity at concentrations 100-fold lower than cyclosporin A, a cyclic undecapeptide that is used to prevent rejection after transplantation of bone marrow and organs, such as kidney, heart, and liver. After the recent discovery that the cyclosporin A-binding protein cyclophilin is identical to peptidylprolyl cis-trans isomerase, a cellular binding protein for FK506 was found to be distinct from cyclophilin but to have the same enzymatic activity. In this study, we isolated a cDNA coding for FK506-binding protein (FKBP) from human peripheral blood T cells by using mixed 20-mer oligonucleotide probes synthesized on the basis of the sequence, Glu-Asp-Gly-Lys-Lys-Phe-Asp, reported for bovine FKBP. The DNA isolated contained an open reading frame encoding 108 amino acid residues. The first 40 residues of the deduced amino acid sequence were identical to those of the reported amino-terminal sequence of bovine FKBP, indicating that the DNA sequence isolated represents the gene coding for FKBP. Computer-assisted analysis of the deduced amino acid sequence indicates that FKBP exhibits no internal homology and does not have significant sequence similarity to any other amino acid sequences of known proteins, including cyclophilin. This result suggests that two catalytically similar proteins, cyclophilin and FKBP, evolved independently. In Northern blot analysis, mRNA species of approximately 1.8 kilobases that hybridized with human FKBP cDNA were detected in poly(A)+ RNAs from brain, lung, liver, and placental cells and leukocytes. Induction of Jurkat leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did not affect the level of FKBP mRNA. Southern blot analysis of human genomic DNA digested with different restriction enzymes suggests the existence of only a few copies of the DNA sequence encoding FKBP. This is in contrast to the result that as many as 20 copies of the cyclophilin gene and possible pseudogenes may be present in the mammalian genome.

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Probing Peptidylprolyl Bond cis/trans Status Using Distal ¹⁹ F NMR Reporters.

Publications

Probing Peptidylprolyl Bond cis/trans Status Using Distal <sup>19</sup> F NMR Reporters.

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Probing Peptidylprolyl Bond cis/trans Status Using Distal 19 F NMR Reporters.

Publications

Probing Peptidylprolyl Bond cis/trans Status Using Distal <sup>19</sup> F NMR Reporters.

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OmicsDI is part of the ELIXIR infrastructure

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Probing Peptidylprolyl Bond cis/trans Status Using Distal ¹⁹ F NMR Reporters.