Photoactive Yellow Protein: From photophysics to materials properties

K.J. Hellingwerf, W. Crielaard, J. Hendriks, Th. Gensch

Laboratory for Microbiology, E.C. Slater Institute, BioCentrum, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands


The photoactive yellow protein (PYP) is a blue-light photoreceptor from the purple-sulphur bacterium Ectothiorhodospira halophila, which shows many similarities with the Archaeal sensory rhodopsins, although PYP contains a 4-hydroxy-cinnamic acid chromophore, and is water-soluble. Activation of PYP proceeds through light-induced trans/cis isomerization of the 7,8-vinyl bond of its chromophore. This configurational transition is followed by a large series of conformational alterations, which ultimately lead to a modulation of the motility machinery of the photoreceptor-producing cell.

PYP, and specific site-directed variants, can be produced heterologously in large amounts as soluble proteins in Escherichia coli. Holoprotein can subsequently be reconstituted with activated coumaryl esters and derivatives thereof. The latter results in the formation of so-called 'hybrid' PYP's.

The coumaryl chromophore of PYP is present in the anionic form in the ground state (pG) of the protein. The anionic phenolate is buried within the hydrophobic core of the protein and is stabilized via a hydrogen-bonding network, involving the amino acids Y42, T50 and (protonated) E46. Photoactivation of PYP generates an excited state that can be observed with fluorescence up-conversion and relaxes multi-exponentially (fastest lifetime: 700 fs) into a photocycle ( = 0.35), containing several transient optical intermediates. At the very short timescale, red-shifted intermediates are formed, which decay into a stable blue-shifted intermediate (pB). In pB, the chromophore is protonated and E46 is deprotonated. As a consequence, at neutral pH no net changes in pH occur when PYP goes through its photocycle: Only intramolecular proton transfer is involved.

This photocycle can be observed in a wide range of pH values, be it that significant effects on the rates of the transitions are observed. Related intermediates can be studied by freeze-trapping. By applying high intensity blue light (~ 450 nm) to a PYP solution pB can be accumulated, the extent of which depends mainly on the pH. The pB-pG photo-equilibrium was used in a number of studies reported in the literature devoted to e.g. Laue diffraction on PYP crystals, solution NMR aimed to resolve the structure of pB, the light induced back-reaction from pB to pG, etc.

The characteristics of the photocycle intermediates of PYP (and of several of its variants, obtained either through site-directed mutagenesis or via reconstitution with non-physiological chromophores) have been investigated with a large range of biophysical techniques. These experiments have led to the conclusion that the characteristics of the conformational transitions in the intermediate states is determined by the mesoscopic environment of the protein.

Significant changes in the pB-to-pG relaxation rate were recently observed in PYP, after establishing a pB-pG photo-equilibrium: A very slowly relaxing sub-population of pB appears (halftime of several 100 s), upon continued illumination at 450 nm. The origin and the nature of this slow-relaxation phase is under investigation, and its consequences for the use of the pB-pG photo-equilibrium as a tool for the investigation of the pB state will be discussed. The photo-stability of PYP against pulsed and continuous visible irradiation has been determined. The results of these experiments will be discussed in the light of literature publications on the materials properties of PYP.