Incoherent Manipulation of the Photoactive Yellow Protein Photocycle with Dispersed Pump-Dump-Probe Spectroscopy

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...

Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Lee

Zewail

2006

“…The earliest structural event, the trans-to-cis photoisomerization of pCA, occurs in ∼1-3 ps (13)(14)(15)(16)(17) and is too fast to track with 150-ps time resolution. Nevertheless, the structural changes arising from this transition are captured in the 100-ps snapshot shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

Schotte

Cho

Kaila

et al. 2012

175

244

To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90°out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/ relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.time-resolved X-ray diffraction | photoreceptor | light sensor

“…Multipulse spectroscopy can be used to control and influence the course of reactions as they evolve, and its power lies in the ability to selectively remove or transfer the population of transient species with carefully timed laser pulses (11)(12)(13). We have recently expanded this technique by combining it with broad-band spectral probing and elaborate global analysis techniques, enabling us to disentangle complex branched reaction schemes (14)(15)(16)(17)(18). In this work, we describe the direct observation of the hidden anionic ground state of GFP as an integral part of the photocycle, revealed through the use of multipulse spectroscopy in combination with global analysis techniques.…”

mentioning

confidence: 99%

Uncovering the hidden ground state of green fluorescent protein

Kennis

Larsen²,

Stokkum³

et al. 2004

Self Cite

140

232

The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I 1 and I2, has been unveiled. I1 and I2 absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I 2 intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen͞deuterium exchange of the protein leads to a significant increase of the I1 and I2 lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.hidden reaction intermediates ͉ multipulse control spectroscopy ͉ proton transfer ͉ ultrafast spectroscopy