Comprehensive Determination of Protein Tyrosine pKa Values for Photoactive Yellow Protein Using Indirect 13C NMR Spectroscopy

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Comprehensive Determination of Protein Tyrosine pKa Values for Photoactive Yellow Protein Using Indirect 13C NMR Spectroscopy

8 February 2012
Publication year: 2012
Source:Biophysical Journal, Volume 102, Issue 3



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 13C ? resonances in sensitive and well-resolved two-dimensional 13C-1H 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 pKa 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.






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