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Substrate-Induced Conformational Changes of the Tyrocidine Synthetase 1 Adenylation Domain Probed by Intrinsic Trp Fluorescence

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Abstract

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA-A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non-cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains.

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Abbreviations

A-domain:

Adenylation domain

T-domain:

Thiolation domain

C-domain:

Condensation domain

TE-domain:

Thioesterase domain

TycA-A:

Tyrocidine synthetase 1 adenylation domain

NRPS:

Nonsibosomal peptide synthetases

ATP:

Adensine triphosphate

PPi :

Pyrophosphate

SrfAC:

Surfactin synthetase

ASA:

Accessible surface area

PheA:

Adenylation domain from gramicidin S synthetase 1

PDB:

Protein data bank

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Acknowledgements

The authors would like to thank Prof. Maja Pavela-Vrančić for providing a financial support through the Croatian Ministry of Science, Education and Sports Grant Number 177-0000000-2962.

Author Contributions

MŠ and VBP designed the experiments, MŠ wrote the manuscript; MŠ and BS performed the experiments; MŠ, SO and VBP analyzed the data.

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Authors and Affiliations

  1. Department of Chemistry, Faculty of Science, University of Split, R. Boškovića 33, HR 21 000, Split, Croatia

    Matilda Šprung, Barbara Soldo, Stjepan Orhanović & Viljemka Bučević-Popović

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  1. Matilda Šprung
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  2. Barbara Soldo
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  3. Stjepan Orhanović
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  4. Viljemka Bučević-Popović
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Correspondence to Matilda Šprung.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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10930_2017_9714_MOESM1_ESM.jpg

Supplementary Fig. 1 The quenching of 227W fluorescence was performed in Tris buffer (50 mM, pH 7.5) supplemented with MgCl2 (20 mM) at 25 °C by adding the aliquots of l-Phe, up to the concentration of 75 µM. (JPG 154 KB)

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Šprung, M., Soldo, B., Orhanović, S. et al. Substrate-Induced Conformational Changes of the Tyrocidine Synthetase 1 Adenylation Domain Probed by Intrinsic Trp Fluorescence. Protein J 36, 202–211 (2017). https://doi.org/10.1007/s10930-017-9714-1

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  • DOI: https://doi.org/10.1007/s10930-017-9714-1

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