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Methionine-121 coordination determines metal specificity in unfolded Pseudomonas aeruginosa azurin

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Abstract

Pseudomonas aeruginosa azurin binds copper so tightly that it remains bound even upon polypeptide unfolding. Copper can be substituted with zinc without change in protein structure, and also in this complex the metal remains bound upon protein unfolding. Previous work has shown that native-state copper ligands Cys112 and His117 are two of at least three metal ligands in the unfolded state. In this study we use isothermal titration calorimetry and spectroscopic methods to test if the native-state ligand Met121 remains a metal ligand upon unfolding. From studies on a point-mutated version of azurin (Met121Ala) and a set of model peptides spanning the copper-binding C-terminal part (including Cys112, His117 and Met121), we conclude that Met121 is a metal ligand in unfolded copper-azurin but not in the case of unfolded zinc-azurin. Combination of unfolding and metal-titration data allow for determination of copper (CuII and CuI) and zinc affinities for folded and unfolded azurin polypeptides, respectively.

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Notes

  1. The CuII form of azurin was selected over the CuI form for several reasons. This is the stable form of copper azurin in air-exposed solutions; therefore redox agents that may obscure far-UV circular dichroism (CD) measurements, are not needed. Visible absorption can be used to monitor unfolding, and most reported data on wild-type and mutant azurins are for CuII forms

Abbreviations

CD:

circular dichroism

GuHCl:

guanidine hydrochloride

ITC:

isothermal titration calorimetry

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Acknowledgements

We thank Andrea Vitello (Tulane University) for help with initial experiments and Dr J. Mierzwa (CIF, Tulane University) for ICP measurements. This work was supported by the National Institutes of Health (GM59663).

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Author notes

  1. Irina Pozdnyakova

    Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

  2. Jesse Guidry

    Present address: Department of Pharmacology, LSU Health Science Center, New Orleans, LA, USA

  3. Pernilla Wittung-Stafshede

    Present address: Department of Biochemistry, Rice University, Houston, TX, USA

Authors and Affiliations

  1. Molecular and Cellular Biology Graduate Program, Tulane University, New Orleans, LA , 70118, USA

    Jessica Marks & Pernilla Wittung-Stafshede

  2. Department of Chemistry, Tulane University, New Orleans, LA , 70118, USA

    Irina Pozdnyakova, Jesse Guidry & Pernilla Wittung-Stafshede

Authors
  1. Jessica Marks
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  2. Irina Pozdnyakova
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  4. Pernilla Wittung-Stafshede
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Correspondence to Pernilla Wittung-Stafshede.

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Marks, J., Pozdnyakova, I., Guidry, J. et al. Methionine-121 coordination determines metal specificity in unfolded Pseudomonas aeruginosa azurin. J Biol Inorg Chem 9, 281–288 (2004). https://doi.org/10.1007/s00775-004-0523-6

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  • DOI: https://doi.org/10.1007/s00775-004-0523-6

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