Chemical Inhomogeneity in the Ultrafast Dynamics of the DXCF

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Chemical Inhomogeneity in the Ultrafast Dynamics of the DXCF Cyanobacteriochrome Tlr0924 Lucy H. Freer, Peter W. Kim, Scott C. Corley, Nathan Clarke Rockwell, Lu Zhao, Arthur Thibert, J. Clark Lagarias, and Delmar S. Larsen J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp302637u • Publication Date (Web): 21 Jun 2012 Downloaded from http://pubs.acs.org on June 26, 2012

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The Journal of Physical Chemistry

Chemical Inhomogeneity in the Ultrafast Dynamics of the DXCF Cyanobacteriochrome Tlr0924

Lucy H. Freer1†, Peter W. Kim1†, Scott C. Corley1, Nathan C. Rockwell2, Lu Zhao1, Arthur J. Thibert1, J. Clark Lagarias2, and Delmar S. Larsen1*

1

Department of Chemistry and 2 Department of Molecular and Cell Biology One Shields Ave, University of California, Davis, 95616

* Corresponding Author: [email protected] †

Lucy H. Freer and Peter W. Kim contributed equally to this paper.

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Abstract Cyanobacteriochromes (CBCRs) are diverse biliprotein photosensors distantly related to the red/far-red photoreceptors of the phytochrome family. There are several subfamilies of CBCRs, displaying diverse spectral responses spanning the entire visible region. Tlr0924 belongs to the DXCF subfamily, which utilizes the Cys residue in a conserved Asp-Xaa-Cys-Phe (DXCF) motif to form a second covalent linkage to the chromophore, resulting in a blue-absorbing dark state. Photoconversion leads to elimination of this linkage, resulting in a green-absorbing photoproduct. Tlr0924 initially incorporates phycocyanobilin (PCB) as chromophore, exhibiting a blue/orange photocycle with PCB, but slowly isomerizes PCB to phycoviolobilin (PVB) to yield the blue/green photocycle. Ultrafast transient absorption spectroscopy was used to study both forward and reverse reaction photodynamics of the recombinant GAF domain of Tlr0924. Primary photoproducts were identified, as were subsequent intermediates at 1 ms. PCB and PVB population photodynamics were decomposed using global target analysis. PCB and PVB populations exhibit similar and parallel photocycles in Tlr0924, but the PVB population exhibits faster excited-state decay in both reaction directions. Based on longer time analysis, our studies show that the photochemical coordinate (15,16-isomerization) and second-linkage coordinate (elimination or bond formation at C10) are separate processes in both directions.


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1. Introduction Phytochromes are well-known red/far-red photosensors first discovered in seed plants1 and later found in cyanobacteria,2 non-oxygenic photosynthetic bacteria,3 heterotrophic bacteria,4 fungi,5 diatoms,6 and algae.7 Phytochromes utilize linear tetrapyrrole (bilin) chromophores to photointerconvert

between

red-absorbing

Pr

and

far-red-absorbing

Pfr

states

via

photoisomerization of the 15,16-double bond of their bilin chromophores (Fig. 1).8-10 Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors that have proliferated in cyanobacterial genomes.9-11 CBCRs and cyanobacterial phytochromes share a highly conserved GAF domain (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) with an invariant Cys residue to which the bilin precursor phycocyanobilin (PCB) is covalently attached.2,12-15 CBCRs lack the phytochrome-specific PHY domain and exhibit more diverse photocycles spanning the entire visible spectrum and the near-UV.16-21 There are several subfamilies of CBCRs.11,20,22 One of these, the DXCF CBCR subfamily, is defined by conserved sequence elements including a conserved Asp-Xaa-Cys-Phe (DXCF) motif containing a second conserved Cys residue.11,21-23 DXCF CBCRs usually exhibit a blueabsorbing dark state, and formation of this state requires the DXCF Cys residue.15,21,22,24 The photoproducts of DXCF CBCRs are more diverse, with examples ranging from blue to orange peak absorbance.21 This diversity is achieved via chemical heterogeneity: while DXCF CBCRs incorporate PCB as chromophore precursor (Fig. 1B), some of them can autocatalytically isomerize it into phycoviolobilin (PVB, Fig. 1A), shortening the conjugated system and generating photoproducts with peak absorbance in the teal or green region of the spectrum.21,24 Other examples retain PCB and exhibit photoproduct absorbance in the yellow to orange region


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of the spectrum, with some containing mixtures of PVB and PCB.21,24 The rate of PVB formation is thus dependent on the protein, and also on the conditions under which the protein is synthesized.21,24 In particular, light exposure can have a major influence on this isomerization because CBCRs such as Tlr0924 from Thermosynechococcus elongatus exhibit pronounced differences in the rate of PVB formation for the two photostates.21 Although PVB formation tunes the absorbance of the photoproduct, it does not have an effect on the blue-absorbing dark state.21,24 This is because the DXCF Cys residue forms a labile covalent linkage to the bilin C10 atom (Fig. 1), effectively creating two conjugated π systems within a single visible light-absorbing bilin chromophore.21,22,24 Photoexcitation triggers isomerization of the bilin 15,16-double bond from the Z configuration to the E configuration, flipping the D-ring within the chromophore binding pocket, followed by thermal elimination of the second linkage on the electronic ground-state surface.21,22,24 After cleavage, PVB and PCB populations have different peak absorption due to their structural differences. Excitation of the photoproduct triggers isomerization of the 15,16-double bond to the Z configuration followed by thermal reformation of the second linkage to complete the two-Cys photocycle.20-22,24 Proteins such as Tlr0924 that contain both PVB and PCB chromophores thus have parallel photocycles for the two populations (Fig. 1). To resolve how this chemical inhomogeneity is manifested in the primary photodynamics of a two-Cys CBCR, we have undertaken the first characterization of the ultrafast photodynamics of the Tlr0924 photocycle in both the forward and reverse reaction directions. The results demonstrate that the PVB and PCB populations follow very similar pathways, but that the PVB population exhibits more rapid excited-state decays in both directions.


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2. Experimental 2.1 Expression and purification Purification of the Tlr0924 CBCR GAF domain as an intein-chitin binding domain (intein-CBD) fusion was performed as described.21 After lysis, ultracentrifugation, and binding to chitin resin (NEB) in accordance with the manufacturer’s instructions, intein cleavage was initiated by addition of DTT to the column, followed by overnight incubation at 4°C. Peak fractions were pooled and dialyzed against TKKG buffer (25 mM TES-KOH pH 7.8, 100 mM KCl, 10% (v/v) glycerol) overnight prior to freezing in liquid nitrogen and storage at -80°C. The resulting preparations had PVB in excess to PCB, as indicated by the major green-absorbing population in the photoproduct spectrum (Fig. 2). As we demonstrated previously,21,25 PVB formation is an ongoing process with a higher rate of PVB formation in the photoproduct state, but it is also a very slow process that competes with other processes including denaturation and thermal reversion to the dark state. Based on the published rates, the change in PVB/PCB composition over the few hours for the ultrafast signals presented below at room temperature is not significant. 2.2 Transient Absorption Signals The dispersed transient absorption setup was constructed from an amplified Ti:sapphire laser system (Spectra Physics Spitfire Pro + Tsunami) operating at 1 kHz, which produced 2.5-mJ pulses of 800-nm fundamental output with a 40-fs (Full Width at Half Maximum) duration. A more detailed description of this setup can be found elsewhere.26 The 800-nm fundamental pulse train from the amplifier was split into multiple paths where one path generated the dispersed white-light probe supercontinuum (350 – 750 nm) by focusing the laser pulses into a slowly translating CaF2 crystal. Two other paths were used to generate the actinic “pump” pulses for the


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forward (blue excitation) and reverse (green excitation) photoreactions. The 450-nm excitation pulses for the forward reaction were generated from the second harmonic of a visible-wavelength home-built non-collinear optical parametric amplifier (NOPA). A 400-nm beam was generated by frequency doubling the 800-nm pulses in a 2-mm thick BBO (β-barium borate) crystal (29.8º) that then pumped a second BBO crystal (32.1º) to generate separate 730-nm signal and 880-nm idler beams. The 880-nm idler beam was frequency doubled within the same BBO crystal to generate the desired 450-nm pump wavelength. The 530-nm excitation pulses used for the reverse kinetics were generated via the standard output of a second independently operating NOPA. The supercontinuum probe beam was optically delayed with respect to the pump pulses by a computer controlled linear-motor stage (Newport IMS 600LMs), which provides up to a 7-ns temporal separation (quadruple pass). The pump pulses were linearly polarized at 54.7o (magic angle) with respect to the probe pulses. The spot size diameters at the sample (300-µm pump and 50-µm probe pulses) were estimated using a micrometer stage and razor blade. The larger pump pulse spot size minimized artifactual contributions to the signals from varying spatial overlap between pump and probe pulses, and was confirmed via observing uniform decay of a transient spectrum when the pump and probe beam spatial overlap was dithered. This indicates that that the probe beam is sufficiently small compared to the pump beam for the measured transient signals to be unaffected by spatial or spectral inhomogeneity of the probe beam. Interrogated Tlr0924 samples were flowed through a two-stage home-built quartz cell setup. The first stage has a 2-mm path length, which allows for both femtosecond measurements (with an OD of ~0.5 per 2 mm at the maximum absorption wavelength) and in situ observation of the


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ground state absorbance spectra to monitor the protein’s condition via a fiber spectrometer (Photon Control PS002). The second cuvette had a 0.1-mm path length and housed two mutually operating LED arrays (Epitex inc. L430-66-60 and L525-66-60) that illuminated the sample to maintain the appropriate state for ultrafast probing. Each LED array was adjusted to produce approximately 10-mW of continuous illumination centered at either 430 nm (for the ultrafast 525-nm induced signals) or 525 nm (for 450-nm ultrafast induced signals). Instrument response functions of approximately 100 fs (both 450-nm and 525-nm pump) were estimated by crosscorrelation of the pump and probe pulses measured by two-photon absorption in buffer solution. Water maintained at 25 ºC was flowed through the aluminum block that housed the two cells to ensure a constant temperature throughout the data collection. 3. Results and Global Analysis Transient absorption spectral data from the forward and reverse photoreactions of Tlr0924 were analyzed using global target analysis methods,27,28 which fit the data to a multipopulation set of interconnected species with fixed spectra and evolution (time) profiles. The application of this analysis approach to the transient pump-probe signals from the red/green NpR6012g4 CBCR can be found elsewhere.29,30 Ultrafast signals were compared to the 1-ms transient absorption spectrum to gain preliminary insight into processes occurring after the 7-ns time range of the ultrafast measurements. 3.1 Forward (15Z → 15E) Ultrafast Photodynamics The transient absorption difference spectra induced by excitation of both PCB and PVB

15Z

Pb

populations exhibit signals across the entire 400-nm spectral probe range (Fig. 3). A negative bleach of the ground-state absorption is observed, as are overlapping excited-state signals. The initial 200-fs transient spectrum (Fig. 3A, black curve) exhibits a broad positive excited-state


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absorption (ESA) from 380 nm to 610 nm with a negative bleach at 445-nm and a negative stimulated emission (SE) band at 500 nm. This spectrum rapidly evolves (

Chemical Inhomogeneity in the Ultrafast Dynamics of the DXCF

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