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Comprehensive determination of protein tyrosine pKa values for photoactive yellow protein using indirect 13C NMR spectroscopy

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  • Nur Alia Oktaviani
  • ,
  • Trijntje J Pool
  • ,
  • Hironari Kamikubo
  • ,
  • Jelle Slager
  • ,
  • Ruud M Scheek
  • ,
  • Mikio Kataoka
  • ,
  • Frans A.A. Mulder
Upon blue-light irradiation, the bacterium Halorhodospira halophila is able to modulate the activity of its flagellar motor and thereby evade potentially harmful UV radiation. The 14 kDa soluble cytosolic photoactive yellow protein (PYP) is believed to be the primary mediator of this photophobic response, and yields a UV/Vis absorption spectrum that closely matches the bacterium's motility spectrum. In the electronic ground state, the para-coumaric acid (pCA) chromophore of PYP is negatively charged and forms two short hydrogen bonds to the side chains of Glu-46 and Tyr-42. The resulting acid triad is central to the marked pH dependence of the optical-absorption relaxation kinetics of PYP. Here, we describe an NMR approach to sequence-specifically follow all tyrosine side-chain protonation states in PYP from pH 3.41 to 11.24. The indirect observation of the nonprotonated (13)C(γ) resonances in sensitive and well-resolved two-dimensional (13)C-(1)H spectra proved to be pivotal in this effort, as observation of other ring-system resonances was hampered by spectral congestion and line-broadening due to ring flips. We observe three classes of tyrosine residues in PYP that exhibit very different pK(a) values depending on whether the phenolic side chain is solvent-exposed, buried, or hydrogen-bonded. In particular, our data show that Tyr-42 remains fully protonated in the pH range of 3.41-11.24, and that pH-induced changes observed in the photocycle kinetics of PYP cannot be caused by changes in the charge state of Tyr-42. It is therefore very unlikely that the pCA chromophore undergoes changes in its electrostatic interactions in the electronic ground state.
Original languageEnglish
JournalBiophysical Journal
Pages (from-to)579-86
Number of pages8
Publication statusPublished - 2012

    Research areas

  • Bacterial Proteins, Crystallography, X-Ray, Halorhodospira halophila, Hydrogen Bonding, Hydrogen-Ion Concentration, Kinetics, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Photoreceptors, Microbial, Physicochemical Processes, Protein Structure, Secondary, Protons, Static Electricity, Tyrosine

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