Primary steps of the photoactive yellow protein: Isolated chromophore dynamics and protein directed function

Abstract

The cycle of the photoactive yellow protein (PYP) has been extensively studied, but the dynamics of the isolated chromophore responsible for transduction is unknown. Here, we present realtime observation of the dynamics of the negatively charged chromophore and detection of intermediates along the path of transto-cis isomerization using femtosecond mass selection͞electron detachment techniques. The results show that the role of the protein environment is not in the first step of double-bond twisting (barrier crossing) but in directing efficient conversion to the cisstructure and in impeding radical formation within the protein.femtobiology ͉ transduction ͉ molecular dynamics ͉ photoelectron spectroscopy P hotoactive yellow protein (PYP) is a water-soluble photoreceptor found in Halorhodospira halophila and related halophilic bacterial species (1). The chromophore responsible for perception of light and phototactic response is a deprotonated trans-4-hydroxycinnamic acid (also known as p-coumaric acid) covalently linked to a cysteine residue of the protein via a thioester bond (Fig. 1). Light absorption of the PYP chromophore leads to the initiation of a complex photocycle involving several intermediates. With a variety of biophysical techniques (2-5), it is concluded that the first intermediate (I 0 ) in the photocycle is formed within a few picoseconds, with the chromophore changing to the cis configuration, in a trans-to-cis isomerization process (7-11). The intermediates at longer times have been trapped and identified by x-ray crystallography (12, 13).The critical change of the chromophore in the primary process of isomerization is accompanied or followed by several other processes: proton transfer, protein conformational change, solvation, and disruption of hydrogen-bonding. This complexity can be reduced if the chromophore in its true anionic, not neutral, structure can be studied free of perturbations. With model compounds, the solvent effect can be assessed by studying them in the solution phase (14-18). However, the nature of intramolecular change and the timescales involved are still unknown. It is, therefore, desirable to study the primary isomerization dynamics of the PYP chromophore in isolation, especially in view of the fact that the mechanism for forming the first intermediate (I 0 ) is debatable (2). A gas-phase spectroscopic study has already been reported (19), but it was for the neutral molecule, not the anion structure in the protein.In this work, we report direct observation of the dynamics of the isolated PYP anionic chromophore. To disentangle the role of protein binding and distant torsions, we preserved the central structure involved in the isomerization about the double bond but use the CH 3 group for termination (see Fig. 1). The chromophore, hereafter called P, is excited with a femtosecond pulse from its ground state, the dark state in the protein, to the trans configuration using the mass-selected ions. For probing we use another femtosecond pulse to photodetach and resolve the phot...

Two Photon‐Induced Electron Injection From a Nanotrigger in Native Endothelial NO‐Synthase

“…[4][5][6] Additional specific interactions of the compounds with protein residues also tune isomerization. [7][8]24] The decay of bound NT* to eNOS was found to be similar with one-and two-photon excitation within experimental error ( Figure 3& Figure 4). The proposed reaction sequence that accounts for decay of NT* bound to eNOS in the presence of the cofactor Ca 2 + /calmodulin is described in Equations (1)- (5):…”

“…This process is strongly dependent on the solvent friction. [7] For example, the shorter excited state lifetime of p-coumaric acid increases with the viscosity. [7,8] This excited state either undergoes a non-radiative transition back to the trans ground state via a ground-state intermediate or decay via the formation of the cis-photoisomer.…”

“…[7] For example, the shorter excited state lifetime of p-coumaric acid increases with the viscosity. [7,8] This excited state either undergoes a non-radiative transition back to the trans ground state via a ground-state intermediate or decay via the formation of the cis-photoisomer. [8,24] In pseudocyanine, trans-stilbene or p-coumaric acid, all carrying the shortest p-conjugated chain, the barrier height to isomerization is either negligible or very low.…”

“…[3][4][5][6] In naturally organized systems as in protein interiors, isomerization may be enhanced by the protein environment compared to homogeneous solution as exemplified by the stilbene-like chromophore of the yellow protein. [7][8] Additionally, photochemical reactions are obviously tuned by the exciting wavelength and two-photon-induced processes depend on the two-photon absorption cross-sections (TPA). [1,9,10] The main and most advantageous use of two photon-induced fluorescence is imaging because it provides high resolution and improved depth penetration in biological samples as well as reduced photodamage.…”

Catching a protein in the act

“…Molecular dynamics simulations have already provided important insights into the early events of PYP photoactivation (19,20). Quite generally, the quality of simulation force fields has much improved.…”