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Ultrafast spectroscopy of biological photoreceptors
Ultrafast spectroscopy of biological photoreceptors John TM Kennis and Marie-Louise Groot We review recent new insights on reaction dynamics of photoreceptors proteins gained from ultrafast spectroscopy. In Blue Light sensing Using FAD (BLUF) domains, a hydrogenbond rearrangement around the flavin chromophore proceeds through a radical-pair mechanism, by which light-induced electron and proton transfer from the protein to flavin result in rotation of a conserved glutamine that switches the hydrogen bond network. Femtosecond infrared spectroscopy has shown that in photoactive yellow protein (PYP), breaking of a hydrogen bond that connects the p-coumaric acid chromophore to the backbone is crucial for trans–cis isomerization and successful entry into the photocycle. Furthermore, isomerization reactions of phycocyanobilin in phytochrome and retinal in the rhodopsins have been revealed in detail through application of femtosecond infrared and femtosecond-stimulated Raman spectroscopy. Addresses Department of Biophysics, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081HV Amsterdam, The Netherlands Corresponding author: Kennis, John TM ([email protected])
Current Opinion in Structural Biology 2007, 17:623–630 This review comes from a themed issue on Biophysical methods Edited by Keith Moffat and Wah Chiu
0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.09.006
Introduction Photoreceptor proteins are exceptionally interesting objects of study, not only in relation to their biological function but also because they can be triggered by a short flash of light, allowing the study of functional protein dynamics over a wide span of timescales. The initial events after photon absorption typically occur in less than 1 ns. As essentially all elementary physical and chemical transformations in biology inherently are ultrafast but often limited by slow diffusional motions that obscure their true dynamics, photoreceptor proteins afford a unique avenue to learn more about the nature of processes such as electron or proton transfer, making or breaking of chemical bonds and motions of small molecular groups. The past decade has witnessed the discovery and characerization of a large number of novel photoreceptors, www.sciencedirect.com
most notably the proteorhodopsins [1], the Light, Oxygen or Voltage (LOV) domains [2–4] and the Blue Light sensing Using FAD (BLUF) domains [5]. BLUF and LOV domains are of special interest as they bind a flavin rather than an isomerizing cofactor, making their photochemistry radically different from that of ‘traditional’ photoreceptors such as the rhodopsins, phytochromes and xanthopsins [6]. The flavin photochemistry enables a strict separation regarding the roles of cofactor and protein: isomerizing cofactors exhibit isomerization and twisting reactions in solution and even in vacuo [7], rendering the influence and catalyzing properties of the protein difficult to assess. By contrast, flavins need partner molecules to react. Thus, flavin-based receptors pose us with new concepts and opportunities to understand how light absorption may be coupled to biological sensory function through efficient and selective photochemistry. While ultrafast spectroscopy at visible and ultraviolet (UV) wavelengths is quite suitable to characterize transient flavin intermediates in LOV and BLUF domains, assessing the structure of isomerizing cofactors while they react remains impossible with this method. This point was explicitly demonstrated in a study on bacteriorhodopsin (bR), where a number of femtosecond optical signals could be well described by a nonreactive model for the retinal chromophore [8]. To determine dynamic structures of chromophores and the interaction with nearby side chains, ultrafast vibrational spectroscopic techniques need to be applied. Here, we review the recent new insights into the early events that occur in biological photoreceptors. We address the photochemistry and the light activation mechanism of BLUF domains and will treat the isomerization mechanisms of photoactive yellow protein, phytochrome and rhodopsins as determined by the novel vibrational techniques, femtosecond infrared (femtoIR) spectroscopy, and femtosecond-stimulated Raman spectroscopy (FSRS).
BLUF domains The flavin cofactor of the BLUF domain, FAD, is noncovalently bound to the protein through a number of hydrogen bonds and hydrophobic interactions. Figure 1A shows the structure of the Rhodobacter sphaeroides AppA BLUF domain in the vicinity of the FAD cofactor in dark and light states [9]. Tyrosine and glutamine side chains are involved in an intricate hydrogen-bond network with flavin. Light absorption results in a hydrogen-bond rearrangement and a red-shift of flavin absorption by 10 nm; the flavin C4 O becomes more strongly hydrogen-bonded [9,10]. This red-shifted state (BLUFRED) probably represents the signaling state of the photoreceptor [11]. Current Opinion in Structural Biology 2007, 17:623–630
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Figure 1
Structure and photocycle dynamics of BLUF domains. (A) X-ray structure of the Rb. sphaeroides AppA BLUF domain in the vicinity of the flavin cofactor in proposed dark state (left) and light state (right) conformations [9]. In the dark, the amino group of conserved glutamine Gln-63 donates hydrogen bonds to flavin N5 and tyrosine Tyr-21. In the light state, Gln-63 has rotated 1808 and its amino group donates a hydrogen bond to flavin C4 O while its carbonyl receives a hydrogen bond from Tyr-21. (B) Kinetic traces of the Synechocystis BLUF domain in H2O (open circles) and D2O (solid circles) recorded upon excitation at 400 nm at selected detection wavelengths; 710 nm denote the decay of FAD singlet-excited state and shows no kinetic isotope effect; 610 nm denote the rise and decay of FAD and FADH radical intermediates, while 483 nm corresponds to the rise of the long-lived product BLUFRED. The latter two probe wavelengths show an obvious kinetic isotope effect, indicating proton or hydrogen movements during these stages of the reaction. (C) Photocycle scheme for photoactivation of BLUF domains along with the time constants for molecular transformations, with those of BLUF in D2O indicated in brackets. The color code corresponds to the spectra of the photocycle intermediates shown in panel (D). Note that FAD* decays with multiple time constants. (D) Absorption difference spectra of the various FAD intermediates of the BLUF photocycle as they follow from kinetic modeling of the time-resolved data.
The photochemical reaction mechanism of BLUF domains was investigated by several groups [12,13, 14–16,17]. Gauden et al. utilized femtosecond transient absorption spectroscopy with multichannel detection, H/ D exchange and extensive kinetic modeling on the Synechocystis and AppA BLUF domains to arrive at the photocycle scheme shown in Figure 1C [12,13]. The spectroscopic signatures of the photocycle intermediates as determined for the Synechocystis BLUF domain are shown in Fig. 1D. The initial light-driven reaction from Current Opinion in Structural Biology 2007, 17:623–630
FAD* involves electron transfer from the protein to result in the FAD– anionic radical, occurring with a dominant time constant of ca. 600 ps in the AppA BLUF domain [12] and much faster (7 ps) in the Synechocystis BLUF domain [13]. The decay of FAD* was highly multiexponential, assigned to different conformational subpopulations of tyrosine (and tryptophan) side chains having variations in the distance to flavin with an ensuing distribution of electron transfer rates [12,13,18]. FAD– is protonated in 6 ps to result in the neutral semiquinone www.sciencedirect.com
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radical FADH. Finally, the light state BLUFRED is formed in 65 ps from FADH. This reaction involves a hydrogen abstraction from FADH. Because FAD* is relatively long lived in the AppA BLUF domain, the FAD– and FADH intermediates did not sufficiently transiently accumulate to be detected. A conserved Trp near FAD (Trp-104 in AppA, Figure 1A) is not required for photocycling and provides a competing nonproductive pathway for light-driven electron and proton transfer [17]. It complements electron/proton transfer to FAD in tyrosine-deficient mutants to result in shortlived FADH–W radical pairs. Mutation of Trp-104 results in an increased yield of BLUFRED, which provides a rare instance of a mutant photoreceptor with a higher product state efficiency than wild type [14]. However, as Trp-104 is probably required to relay photon detection to the molecular surface [9], Trp-104 mutants do not transduce signals in vivo [19]. Removal of both Tyr and Trp completely abolishes redox photochemistry: FAD* then only produces triplets through intersystem crossing. Figure 2 shows a detailed reaction mechanism for BLUF photoactivation [13]. With the hydrogen-bond rearrangement around FAD complete in less than 1 ns, slower yet unknown conformational changes probably take place that affect the BLUF molecular surface and its interaction with intramolecular or intermolecular signaling partners. The
BLUF photochemistry is distinct from that of LOV domains, where the flavin-excited state is photochemically inert and only undergoes intersystem crossing to the flavin triplet state in a few nanoseconds [20,21]. From the triplet state, a covalent bond is formed between flavin and a cysteine on the microsecond timescale [4]. Remarkably, photolysis of the covalent flavin–cysteinyl bond with near-UV light results in regeneration of dark-state LOV [22].
PYP The vibrational spectrum of a protein or a protein-bound chromophore contains a wealth of information about its structure, the interaction with the environment and electronic properties. Time-resolved IR spectroscopy is a powerful tool that can reveal many of the dynamic structural details of chromophores involved in (photo)biological reactions [23,24]. In addition, it can reveal the response of those parts of the protein that are affected by the ongoing reactions. Following infrared absorption changes in real time offer the exciting possibility to observe real-time dynamic changes of the chromophore and protein residues and relate those, with the help of Xray diffraction structures, mutations, isotope labeling and normal mode calculations, to physical reaction mechanisms and protein structural changes. The chromophore in PYP is a negatively charged p-coumaric acid that is covalently bound to the protein
Figure 2
Hydrogen-bond switch model for BLUF photoactivation [13]. Light absorption by FAD drives electron transfer from Tyr with multiple time constants between 7 and 600 ps to result in FAD– and Tyr+ that breaks the hydrogen bond between Tyr and Gln because of electrostatic repulsion. Then, proton transfer from Tyr to FAD– occurs in 6 ps, leading to FADH–Tyr radical pairs. Protonation takes place at FAD N5, breaking the hydrogen bond from Gln to FAD. These events leave Gln unhinged and allow its 1808 rotation, resulting in a strong hydrogen bond from the amino moiety of Gln to C4 O of FADH. Then radical-pair recombination follows in 65 ps that re-oxidizes FAD and locks the glutamine in place by accepting a hydrogen bond from Tyr to its carbonyl moiety. www.sciencedirect.com
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Figure 3
(A) Schematic drawing of the active site of dark-state wild-type PYP. The all-trans p-coumaric acid chromophore is covalently bound to the protein backbone through Cys-69, and its carbonyl is hydrogen-bonded to Cys-69. The negatively charged phenol moiety is hydrogen bonded to Gln-46 and Tyr-42. (B) Conformation of p-coumaric acid in the I1 intermediate state with an isomerized chromophore, 1808 rotation of its carbonyl, and a broken hydrogen bond to Cys-69.
through a thioester linkage (Figure 3A) Upon absorption of light, PYP enters a photocycle involving a trans/cis isomerization, followed by chromophore protonation and partial protein unfolding into the signaling state [25]. Several ultrafast transient absorption studies have been devoted on the initial dynamics [26–29]. The excited state decays in a few picoseconds into the first intermediate I0 and after 1 ns the next intermediate I1 is formed that corresponds to a cis-isomer (Figure 3B) [25]. Figure 4C shows the vibrational absorption difference spectra of I0 and I1 obtained by femtoIR spectroscopy [30,31]. Figure 4A shows representative time traces, together with a fit to the data with three exponential decays of 2, 800 ps and a long-lived component. The first component results from excited-state (ES) decay and the formation of I0 at a yield of 30%. I1 is formed after 800 ps. The close spectral resemblance of I0 and I1 (Figure 4C, red and blue lines respectively) indicates that I0 is an unrelaxed cis-isomer, as judged from trans–cis markers near 1300 and 1330 cm 1 (bleached trans bands) and 1290 cm 1 (up going cis band) [30]. Also, the C O vibration of the chromophore up shifts from 1650 to 1670 cm 1, indicating that the hydrogen bond to Cys-69 is broken. Thus, the cis-isomer ground state in PYP appears in 2 ps. Heyne et al. provided further support for these assignments through observation of additional isomerization marker modes and quantum-chemical calculations [32]. In the E46Q mutant, Glu-46 that donates a hydrogen bond to the phenol ring of the p-coumaric acid in wildtype PYP is replaced by a weaker hydrogen bond donating glutamine (Gln) that also cannot donate a proton to the phenolate oxygen. The C O frequency of Gln-46 shifts from 1685 cm 1 in the ground state to 1697 cm 1 in the excited states and I0 states. In I1 it further shifts to 1704 cm 1 (Figure 4C). Thus, this hydrogen bond breaks during the latter transition in 800 ps. In WT PYP the Current Opinion in Structural Biology 2007, 17:623–630
hydrogen bond between Glu-46 and the chromophore is strengthened on this timescale [30]. A major fraction of initially excited chromophores (70%) relaxes back to the original all-trans ground state. Pump– dump–probe experiments in the visible showed that these chromophores proceed through a distinct groundstate intermediate (GSI) that has a lifetime of 6 ps (Figure 4B) [29]. FemtoIR spectroscopy indicated that with its trans–cis markers near 1300 cm 1 (Figure 4C, green line), the GSI species had a cis-like structure similar to that of I0 and I1. Remarkably, however, it retained its hydrogen bond with Cys-69 as judged from the absence of the chromophore C O signal at 1650 cm 1. The sequence of events that occurs in PYP upon excitation can now be constructed as follows [31,33]. In the excited state, the negative charge on the phenolic oxygen migrates toward the thio-ester linkage [34], giving the double bond of the hydrogen-bonded carbonyl a singlebond character. The hydrogen bond to Cys-69 remains initially intact. Isomerization proceeds partially in the excited state and continues in the ground state, where the chromophore follows one of two possible pathways: (i) it breaks its hydrogen bond and forms I0 or (ii) the hydrogen bond remains intact, and the chromophore reisomerizes to the ground state. The isomerization process and the breaking of the hydrogen bond are therefore independent processes, and the strength of the hydrogen bond with Cys-69 determines whether the chromophore will enter the photocycle. The hydrogen bond of the chromophore phenolic ring with Glu-46 does not seem to play a crucial role in the initial part of the photocycle.
Phytochromes A detailed understanding of the phytochrome photoactivation mechanism was long hampered by a lack of structural information. This constraint was largely solved with www.sciencedirect.com
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Figure 4
Ultrafast dynamics of PYP and its E46Q mutant as measured with femtoIR spectroscopy [31]. (A) Selection of kinetic traces taken on the E46Q mutant of PYP with femtoIR spectroscopy. The black lines correspond to fits to the data based on the kinetic model shown in panel (B). (B) Kinetic model used to describe the ultrafast light induced dynamics of PYP. ES corresponds to the excited state of p-coumaric acid, GSI to a ground-state intermediate that decays to the original all-trans ground state. I0 and I1 correspond to the first photocycle intermediates of PYP. (C) Absorption difference spectra of the various molecular states in the kinetic model of panel (B) of wild-type PYP and the E46Q mutant, with the ES spectra are shown in a, GSI in b, I0 and I1 in c and d. Note that c and d share the same legend. Negative features in these spectra originate from the ground-state bleach, and positive ones originate from ES or product state absorption.
the X-ray structures of the biliverdin-binding domains from Deinococcus radiodurans and Rhodopseudomonas palustris bacteriophytochrome [35,36]. A femtoIR study on cyanobacterial phytochrome Cph1 indicated that the primary intermediate Lumi-R was generated from the phycocyanobilin excited state with multiple time constants of 3, 14, and 134 ps at a quantum yield of approximately 10%. Lumi-R exhibited a downshift of C C and C O modes that were consistent with C15 Z/E isomerization, corresponding to rotation of ring IV. Unexpectwww.sciencedirect.com
edly, torsional angle changes at C5, located between rings I and II were observed as well. Importantly, the infrared signature of Lumi-R was significantly different from that observed with cryotrapping-FTIR [37].
Rhodopsins As the rhodopsins have been prominently at the forefront of photoreceptor research for decades now, they have been the first candidates for application of ultrafast vibrational spectroscopy [38,39]. The advent of FSRS has been a major Current Opinion in Structural Biology 2007, 17:623–630
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recent development in this regard. FSRS is a three-pulse Raman technique that combines a high time resolution (<100 fs) with high spectral resolution (<15 cm 1). An excitation pulse that initiates the photochemistry is followed by a pulse pair overlapped in time comprising a narrowband picosecond Raman pump pulse and a femtosecond white light continuum probe pulse. Through the third order nonlinear susceptibility, the white light probe is amplified at Raman-active frequencies, and a Raman spectrum is imprinted on the white light. A full account of the technique is given in reference [40]. The isomerization mechanism of 11-cis retinal in rhodopsin was unveiled in unprecedented detail by FSRS [41]. Isomerization to all-trans retinal was previously shown to occur in less than 200 fs [42]. The FSRS experiments confirmed that within this exceedingly short time (corresponding to the photorhodopsin intermediate), the C11 C12 double bond had indeed formally isomerized, most probably along impulsively excited hydrogen-outof-plane (HOOP) wagging modes. However, retinal still largely retained its dark-state structure after 200 fs, and relaxation to a strained all-trans conformation (the bathorhodopsin intermediate) took place in 1 ps on the newly populated ground-state potential energy surface. Application of FSRS to bacteriorhodopsin was markedly less successful as the FSRS signals were dominated by ground-state Raman features during the first picosecond caused by resonance of the 800 nm Raman pump with stimulated emission from retinal excited state [43]. Remarkably, these signals decayed significantly faster (250 fs) than the stimulated emission, which was interpreted as dissipation of Franck-Condon (FC) active modes into FC-silent reactive modes, primarily HOOPS and torsions that were proposed to further drive the isomerization reaction. Ultrafast transient absorption spectroscopy on proteorhodopsin revealed that the timescales and spectroscopic signatures of retinal all-trans to 13-cis isomerization were very similar to those of bacteriorhodopsin [44]. A hotly debated issue concerns the application of impulsive vibrational spectroscopy (IVS) to bR. Here, an extremely short optical pulse of 5–7 fs is used to impulsively excite all FC-active vibrational modes of retinal, including high-frequency C C and C–C stretches in the 1500–1700 cm 1 region. An equally short pulse is used as a probe and detected in a wavelength-resolved fashion, revealing strong oscillatory patterns as a function of time. Fourier transformation in various forms may then be applied to extract the (dynamic) mode frequencies. In this way, detailed stretching and torsional motions in the retinal-excited state before isomerization were monitored [38]. However, already in the nineties such oscillatory patterns were ascribed to an impulsive stimulated Raman process that populated wavepackets on the ground-state Current Opinion in Structural Biology 2007, 17:623–630
potential energy surface [45]. By applying chirped excitation pulses it was recently demonstrated that the IVS signals indeed mainly corresponded to ground-state wavepacket motion [46]. Only by detection at the short-wavelength side of the bR absorption (which coincides with an intense excited-state absorption of retinal) could the signals tentatively be assigned to motion on the excited-state potential energy surface. Moreover, it was observed that previously applied sliding window Fourier transform led to conflicts in time resolution and frequency resolution. Thus, the interpretation of IVS remains cumbersome for now.
Acknowledgements The authors are indebted to Magdalena Gauden, Rienk van Grondelle, Peter Hegemann, Klaas Hellingwerf, Ivo van Stokkum and Luuk van Wilderen for their contributions to some of the work reviewed in this paper. JTMK and MLG were supported by the Life Sciences Council of the Netherlands Organization for Scientific Research (NWO-ALW) through VIDI fellowships.
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twisting around the C5 bond was observed at a previously considered structurally inactive part of the chromophore. 38. Kobayashi T, Saito T, Ohtani H: Real-time spectroscopy of transition states in bacteriorhodopsin during retinal isomerization. Nature 2001, 414:531-534. 39. Herbst J, Heyne K, Diller R: Femtosecond infrared spectroscopy of bacteriorhodopsin chromophore isomerization. Science 2002, 297:822-825. 40. Kukura P, McCamant DW, Mathies RA: Femtosecond stimulated raman spectroscopy. Annu Rev Phys Chem 2007, 58:461-488. This review provides a comprehensive overview of femtosecond-stimulated Raman spectroscopy and its application to various photoactive systems. 41. Kukura P, McCamant DW, Yoon S, Wandschneider DB, Mathies RA: Structural observation of the primary isomerization in vision with femtosecond-stimulated Raman. Science 2005, 310:1006-1009. The isomerization mechanism of rhodopsin was revealed in unprecedented detail with femtosecond-stimulated Raman spectroscopy. Isomerization around the C11 C12 double bond was found to occur in less than 200 fs. Further evolution to a strained all-trans structure took place in 1 ps. 42. Schoenlein RW, Peteanu LA, Mathies RA, Shank CV: The 1st step in vision—femtosecond isomerization of Rhodopsin. Science 1991, 254:412-415.
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43. McCamant DW, Kukura P, Mathies RA: Femtosecond stimulated Raman study of excited-state evolution in bacteriorhodopsin. J Phys Chem B 2005, 109:10449-10457. This paper concerns a femtosecond-stimulated Raman study on bacteriorhodopsin. Although no dynamic structural information is obtained during the first picosecond because of distortion of the signals by ground-state Raman features, evidence is provided for dissipation of Franck-Condon active modes of retinal into reactive low-frequency modes. 44. Lenz MO, Huber R, Schmidt B, Gilch P, Kalmbach R, Engelhard M, Wachtveitl J: First steps of retinal photoisomerization in proteorhodopsin. Biophys J 2006, 91:255-262. Here, a first full account of the excited-state reactions of retinal in proteorhodopsin is given as probed with visible ultrafast spectroscopy. Isomerization of all-trans retinal was found to proceed in a way similar to that of bacteriorhodopsin. 45. Pollard WT, Mathies RA: Analysis of femtosecond dynamic absorption spectra of nonstationary states. Annu Rev Phys Chem 1992, 43:497-523. 46. Kahan A, Nahmias O, Friedman N, Sheves M, Ruhman S: Following photoinduced dynamics in bacteriorhodopsin with 7-fs impulsive vibrational spectroscopy. J Am Chem Soc 2007, 129:537-546. This paper concerns a comprehensive study of impulsive vibrational spectroscopy (IVS) on bacteriorhodopsin. It is demonstrated that the IVS signals mainly arise from an impulsive stimulated Raman process creating wavepackets on the ground-state potential energy surface, and thus limited information on excited-state dynamics is obtained.
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Current Opinion in Structural Biology 2007, 17:623–630 This review comes from a themed issue on Biophysical methods Edited by Keith Moffat and Wah Chiu
0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.09.006
Introduction Photoreceptor proteins are exceptionally interesting objects of study, not only in relation to their biological function but also because they can be triggered by a short flash of light, allowing the study of functional protein dynamics over a wide span of timescales. The initial events after photon absorption typically occur in less than 1 ns. As essentially all elementary physical and chemical transformations in biology inherently are ultrafast but often limited by slow diffusional motions that obscure their true dynamics, photoreceptor proteins afford a unique avenue to learn more about the nature of processes such as electron or proton transfer, making or breaking of chemical bonds and motions of small molecular groups. The past decade has witnessed the discovery and characerization of a large number of novel photoreceptors, www.sciencedirect.com
most notably the proteorhodopsins [1], the Light, Oxygen or Voltage (LOV) domains [2–4] and the Blue Light sensing Using FAD (BLUF) domains [5]. BLUF and LOV domains are of special interest as they bind a flavin rather than an isomerizing cofactor, making their photochemistry radically different from that of ‘traditional’ photoreceptors such as the rhodopsins, phytochromes and xanthopsins [6]. The flavin photochemistry enables a strict separation regarding the roles of cofactor and protein: isomerizing cofactors exhibit isomerization and twisting reactions in solution and even in vacuo [7], rendering the influence and catalyzing properties of the protein difficult to assess. By contrast, flavins need partner molecules to react. Thus, flavin-based receptors pose us with new concepts and opportunities to understand how light absorption may be coupled to biological sensory function through efficient and selective photochemistry. While ultrafast spectroscopy at visible and ultraviolet (UV) wavelengths is quite suitable to characterize transient flavin intermediates in LOV and BLUF domains, assessing the structure of isomerizing cofactors while they react remains impossible with this method. This point was explicitly demonstrated in a study on bacteriorhodopsin (bR), where a number of femtosecond optical signals could be well described by a nonreactive model for the retinal chromophore [8]. To determine dynamic structures of chromophores and the interaction with nearby side chains, ultrafast vibrational spectroscopic techniques need to be applied. Here, we review the recent new insights into the early events that occur in biological photoreceptors. We address the photochemistry and the light activation mechanism of BLUF domains and will treat the isomerization mechanisms of photoactive yellow protein, phytochrome and rhodopsins as determined by the novel vibrational techniques, femtosecond infrared (femtoIR) spectroscopy, and femtosecond-stimulated Raman spectroscopy (FSRS).
BLUF domains The flavin cofactor of the BLUF domain, FAD, is noncovalently bound to the protein through a number of hydrogen bonds and hydrophobic interactions. Figure 1A shows the structure of the Rhodobacter sphaeroides AppA BLUF domain in the vicinity of the FAD cofactor in dark and light states [9]. Tyrosine and glutamine side chains are involved in an intricate hydrogen-bond network with flavin. Light absorption results in a hydrogen-bond rearrangement and a red-shift of flavin absorption by 10 nm; the flavin C4 O becomes more strongly hydrogen-bonded [9,10]. This red-shifted state (BLUFRED) probably represents the signaling state of the photoreceptor [11]. Current Opinion in Structural Biology 2007, 17:623–630
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Figure 1
Structure and photocycle dynamics of BLUF domains. (A) X-ray structure of the Rb. sphaeroides AppA BLUF domain in the vicinity of the flavin cofactor in proposed dark state (left) and light state (right) conformations [9]. In the dark, the amino group of conserved glutamine Gln-63 donates hydrogen bonds to flavin N5 and tyrosine Tyr-21. In the light state, Gln-63 has rotated 1808 and its amino group donates a hydrogen bond to flavin C4 O while its carbonyl receives a hydrogen bond from Tyr-21. (B) Kinetic traces of the Synechocystis BLUF domain in H2O (open circles) and D2O (solid circles) recorded upon excitation at 400 nm at selected detection wavelengths; 710 nm denote the decay of FAD singlet-excited state and shows no kinetic isotope effect; 610 nm denote the rise and decay of FAD and FADH radical intermediates, while 483 nm corresponds to the rise of the long-lived product BLUFRED. The latter two probe wavelengths show an obvious kinetic isotope effect, indicating proton or hydrogen movements during these stages of the reaction. (C) Photocycle scheme for photoactivation of BLUF domains along with the time constants for molecular transformations, with those of BLUF in D2O indicated in brackets. The color code corresponds to the spectra of the photocycle intermediates shown in panel (D). Note that FAD* decays with multiple time constants. (D) Absorption difference spectra of the various FAD intermediates of the BLUF photocycle as they follow from kinetic modeling of the time-resolved data.
The photochemical reaction mechanism of BLUF domains was investigated by several groups [12,13, 14–16,17]. Gauden et al. utilized femtosecond transient absorption spectroscopy with multichannel detection, H/ D exchange and extensive kinetic modeling on the Synechocystis and AppA BLUF domains to arrive at the photocycle scheme shown in Figure 1C [12,13]. The spectroscopic signatures of the photocycle intermediates as determined for the Synechocystis BLUF domain are shown in Fig. 1D. The initial light-driven reaction from Current Opinion in Structural Biology 2007, 17:623–630
FAD* involves electron transfer from the protein to result in the FAD– anionic radical, occurring with a dominant time constant of ca. 600 ps in the AppA BLUF domain [12] and much faster (7 ps) in the Synechocystis BLUF domain [13]. The decay of FAD* was highly multiexponential, assigned to different conformational subpopulations of tyrosine (and tryptophan) side chains having variations in the distance to flavin with an ensuing distribution of electron transfer rates [12,13,18]. FAD– is protonated in 6 ps to result in the neutral semiquinone www.sciencedirect.com
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radical FADH. Finally, the light state BLUFRED is formed in 65 ps from FADH. This reaction involves a hydrogen abstraction from FADH. Because FAD* is relatively long lived in the AppA BLUF domain, the FAD– and FADH intermediates did not sufficiently transiently accumulate to be detected. A conserved Trp near FAD (Trp-104 in AppA, Figure 1A) is not required for photocycling and provides a competing nonproductive pathway for light-driven electron and proton transfer [17]. It complements electron/proton transfer to FAD in tyrosine-deficient mutants to result in shortlived FADH–W radical pairs. Mutation of Trp-104 results in an increased yield of BLUFRED, which provides a rare instance of a mutant photoreceptor with a higher product state efficiency than wild type [14]. However, as Trp-104 is probably required to relay photon detection to the molecular surface [9], Trp-104 mutants do not transduce signals in vivo [19]. Removal of both Tyr and Trp completely abolishes redox photochemistry: FAD* then only produces triplets through intersystem crossing. Figure 2 shows a detailed reaction mechanism for BLUF photoactivation [13]. With the hydrogen-bond rearrangement around FAD complete in less than 1 ns, slower yet unknown conformational changes probably take place that affect the BLUF molecular surface and its interaction with intramolecular or intermolecular signaling partners. The
BLUF photochemistry is distinct from that of LOV domains, where the flavin-excited state is photochemically inert and only undergoes intersystem crossing to the flavin triplet state in a few nanoseconds [20,21]. From the triplet state, a covalent bond is formed between flavin and a cysteine on the microsecond timescale [4]. Remarkably, photolysis of the covalent flavin–cysteinyl bond with near-UV light results in regeneration of dark-state LOV [22].
PYP The vibrational spectrum of a protein or a protein-bound chromophore contains a wealth of information about its structure, the interaction with the environment and electronic properties. Time-resolved IR spectroscopy is a powerful tool that can reveal many of the dynamic structural details of chromophores involved in (photo)biological reactions [23,24]. In addition, it can reveal the response of those parts of the protein that are affected by the ongoing reactions. Following infrared absorption changes in real time offer the exciting possibility to observe real-time dynamic changes of the chromophore and protein residues and relate those, with the help of Xray diffraction structures, mutations, isotope labeling and normal mode calculations, to physical reaction mechanisms and protein structural changes. The chromophore in PYP is a negatively charged p-coumaric acid that is covalently bound to the protein
Figure 2
Hydrogen-bond switch model for BLUF photoactivation [13]. Light absorption by FAD drives electron transfer from Tyr with multiple time constants between 7 and 600 ps to result in FAD– and Tyr+ that breaks the hydrogen bond between Tyr and Gln because of electrostatic repulsion. Then, proton transfer from Tyr to FAD– occurs in 6 ps, leading to FADH–Tyr radical pairs. Protonation takes place at FAD N5, breaking the hydrogen bond from Gln to FAD. These events leave Gln unhinged and allow its 1808 rotation, resulting in a strong hydrogen bond from the amino moiety of Gln to C4 O of FADH. Then radical-pair recombination follows in 65 ps that re-oxidizes FAD and locks the glutamine in place by accepting a hydrogen bond from Tyr to its carbonyl moiety. www.sciencedirect.com
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Figure 3
(A) Schematic drawing of the active site of dark-state wild-type PYP. The all-trans p-coumaric acid chromophore is covalently bound to the protein backbone through Cys-69, and its carbonyl is hydrogen-bonded to Cys-69. The negatively charged phenol moiety is hydrogen bonded to Gln-46 and Tyr-42. (B) Conformation of p-coumaric acid in the I1 intermediate state with an isomerized chromophore, 1808 rotation of its carbonyl, and a broken hydrogen bond to Cys-69.
through a thioester linkage (Figure 3A) Upon absorption of light, PYP enters a photocycle involving a trans/cis isomerization, followed by chromophore protonation and partial protein unfolding into the signaling state [25]. Several ultrafast transient absorption studies have been devoted on the initial dynamics [26–29]. The excited state decays in a few picoseconds into the first intermediate I0 and after 1 ns the next intermediate I1 is formed that corresponds to a cis-isomer (Figure 3B) [25]. Figure 4C shows the vibrational absorption difference spectra of I0 and I1 obtained by femtoIR spectroscopy [30,31]. Figure 4A shows representative time traces, together with a fit to the data with three exponential decays of 2, 800 ps and a long-lived component. The first component results from excited-state (ES) decay and the formation of I0 at a yield of 30%. I1 is formed after 800 ps. The close spectral resemblance of I0 and I1 (Figure 4C, red and blue lines respectively) indicates that I0 is an unrelaxed cis-isomer, as judged from trans–cis markers near 1300 and 1330 cm 1 (bleached trans bands) and 1290 cm 1 (up going cis band) [30]. Also, the C O vibration of the chromophore up shifts from 1650 to 1670 cm 1, indicating that the hydrogen bond to Cys-69 is broken. Thus, the cis-isomer ground state in PYP appears in 2 ps. Heyne et al. provided further support for these assignments through observation of additional isomerization marker modes and quantum-chemical calculations [32]. In the E46Q mutant, Glu-46 that donates a hydrogen bond to the phenol ring of the p-coumaric acid in wildtype PYP is replaced by a weaker hydrogen bond donating glutamine (Gln) that also cannot donate a proton to the phenolate oxygen. The C O frequency of Gln-46 shifts from 1685 cm 1 in the ground state to 1697 cm 1 in the excited states and I0 states. In I1 it further shifts to 1704 cm 1 (Figure 4C). Thus, this hydrogen bond breaks during the latter transition in 800 ps. In WT PYP the Current Opinion in Structural Biology 2007, 17:623–630
hydrogen bond between Glu-46 and the chromophore is strengthened on this timescale [30]. A major fraction of initially excited chromophores (70%) relaxes back to the original all-trans ground state. Pump– dump–probe experiments in the visible showed that these chromophores proceed through a distinct groundstate intermediate (GSI) that has a lifetime of 6 ps (Figure 4B) [29]. FemtoIR spectroscopy indicated that with its trans–cis markers near 1300 cm 1 (Figure 4C, green line), the GSI species had a cis-like structure similar to that of I0 and I1. Remarkably, however, it retained its hydrogen bond with Cys-69 as judged from the absence of the chromophore C O signal at 1650 cm 1. The sequence of events that occurs in PYP upon excitation can now be constructed as follows [31,33]. In the excited state, the negative charge on the phenolic oxygen migrates toward the thio-ester linkage [34], giving the double bond of the hydrogen-bonded carbonyl a singlebond character. The hydrogen bond to Cys-69 remains initially intact. Isomerization proceeds partially in the excited state and continues in the ground state, where the chromophore follows one of two possible pathways: (i) it breaks its hydrogen bond and forms I0 or (ii) the hydrogen bond remains intact, and the chromophore reisomerizes to the ground state. The isomerization process and the breaking of the hydrogen bond are therefore independent processes, and the strength of the hydrogen bond with Cys-69 determines whether the chromophore will enter the photocycle. The hydrogen bond of the chromophore phenolic ring with Glu-46 does not seem to play a crucial role in the initial part of the photocycle.
Phytochromes A detailed understanding of the phytochrome photoactivation mechanism was long hampered by a lack of structural information. This constraint was largely solved with www.sciencedirect.com
Ultrafast spectroscopy of biological photoreceptors Kennis and Groot 627
Figure 4
Ultrafast dynamics of PYP and its E46Q mutant as measured with femtoIR spectroscopy [31]. (A) Selection of kinetic traces taken on the E46Q mutant of PYP with femtoIR spectroscopy. The black lines correspond to fits to the data based on the kinetic model shown in panel (B). (B) Kinetic model used to describe the ultrafast light induced dynamics of PYP. ES corresponds to the excited state of p-coumaric acid, GSI to a ground-state intermediate that decays to the original all-trans ground state. I0 and I1 correspond to the first photocycle intermediates of PYP. (C) Absorption difference spectra of the various molecular states in the kinetic model of panel (B) of wild-type PYP and the E46Q mutant, with the ES spectra are shown in a, GSI in b, I0 and I1 in c and d. Note that c and d share the same legend. Negative features in these spectra originate from the ground-state bleach, and positive ones originate from ES or product state absorption.
the X-ray structures of the biliverdin-binding domains from Deinococcus radiodurans and Rhodopseudomonas palustris bacteriophytochrome [35,36]. A femtoIR study on cyanobacterial phytochrome Cph1 indicated that the primary intermediate Lumi-R was generated from the phycocyanobilin excited state with multiple time constants of 3, 14, and 134 ps at a quantum yield of approximately 10%. Lumi-R exhibited a downshift of C C and C O modes that were consistent with C15 Z/E isomerization, corresponding to rotation of ring IV. Unexpectwww.sciencedirect.com
edly, torsional angle changes at C5, located between rings I and II were observed as well. Importantly, the infrared signature of Lumi-R was significantly different from that observed with cryotrapping-FTIR [37].
Rhodopsins As the rhodopsins have been prominently at the forefront of photoreceptor research for decades now, they have been the first candidates for application of ultrafast vibrational spectroscopy [38,39]. The advent of FSRS has been a major Current Opinion in Structural Biology 2007, 17:623–630
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recent development in this regard. FSRS is a three-pulse Raman technique that combines a high time resolution (<100 fs) with high spectral resolution (<15 cm 1). An excitation pulse that initiates the photochemistry is followed by a pulse pair overlapped in time comprising a narrowband picosecond Raman pump pulse and a femtosecond white light continuum probe pulse. Through the third order nonlinear susceptibility, the white light probe is amplified at Raman-active frequencies, and a Raman spectrum is imprinted on the white light. A full account of the technique is given in reference [40]. The isomerization mechanism of 11-cis retinal in rhodopsin was unveiled in unprecedented detail by FSRS [41]. Isomerization to all-trans retinal was previously shown to occur in less than 200 fs [42]. The FSRS experiments confirmed that within this exceedingly short time (corresponding to the photorhodopsin intermediate), the C11 C12 double bond had indeed formally isomerized, most probably along impulsively excited hydrogen-outof-plane (HOOP) wagging modes. However, retinal still largely retained its dark-state structure after 200 fs, and relaxation to a strained all-trans conformation (the bathorhodopsin intermediate) took place in 1 ps on the newly populated ground-state potential energy surface. Application of FSRS to bacteriorhodopsin was markedly less successful as the FSRS signals were dominated by ground-state Raman features during the first picosecond caused by resonance of the 800 nm Raman pump with stimulated emission from retinal excited state [43]. Remarkably, these signals decayed significantly faster (250 fs) than the stimulated emission, which was interpreted as dissipation of Franck-Condon (FC) active modes into FC-silent reactive modes, primarily HOOPS and torsions that were proposed to further drive the isomerization reaction. Ultrafast transient absorption spectroscopy on proteorhodopsin revealed that the timescales and spectroscopic signatures of retinal all-trans to 13-cis isomerization were very similar to those of bacteriorhodopsin [44]. A hotly debated issue concerns the application of impulsive vibrational spectroscopy (IVS) to bR. Here, an extremely short optical pulse of 5–7 fs is used to impulsively excite all FC-active vibrational modes of retinal, including high-frequency C C and C–C stretches in the 1500–1700 cm 1 region. An equally short pulse is used as a probe and detected in a wavelength-resolved fashion, revealing strong oscillatory patterns as a function of time. Fourier transformation in various forms may then be applied to extract the (dynamic) mode frequencies. In this way, detailed stretching and torsional motions in the retinal-excited state before isomerization were monitored [38]. However, already in the nineties such oscillatory patterns were ascribed to an impulsive stimulated Raman process that populated wavepackets on the ground-state Current Opinion in Structural Biology 2007, 17:623–630
potential energy surface [45]. By applying chirped excitation pulses it was recently demonstrated that the IVS signals indeed mainly corresponded to ground-state wavepacket motion [46]. Only by detection at the short-wavelength side of the bR absorption (which coincides with an intense excited-state absorption of retinal) could the signals tentatively be assigned to motion on the excited-state potential energy surface. Moreover, it was observed that previously applied sliding window Fourier transform led to conflicts in time resolution and frequency resolution. Thus, the interpretation of IVS remains cumbersome for now.
Acknowledgements The authors are indebted to Magdalena Gauden, Rienk van Grondelle, Peter Hegemann, Klaas Hellingwerf, Ivo van Stokkum and Luuk van Wilderen for their contributions to some of the work reviewed in this paper. JTMK and MLG were supported by the Life Sciences Council of the Netherlands Organization for Scientific Research (NWO-ALW) through VIDI fellowships.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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10. Masuda S, Hasegawa K, Ishii A, Ono T: Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of Synechocystis sp PCC6803. Biochemistry 2004, 43:5304-5313. www.sciencedirect.com
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11. Masuda S, Bauer CE: AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell 2002, 110:613-623. 12. Gauden M, Yeremenko S, Laan W, van Stokkum IHM, Ihalainen JA, van Grondelle R, Hellingwerf KJ, Kennis JTM: Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of photosynthesis genes. Biochemistry 2005, 44:3653-3662. The first full characterization of the early stages of the BLUF photocycle is reported from femtoseconds to milliseconds. It is shown that the redshifted product state is formed in less than 1 ns and that no further spectral changes in the UV–vis occur up to milliseconds. A minor fraction of flavin triplet states was shown to represent a physiologically irrelevant side reaction. 13. Gauden M, van Stokkum IHM, Key JM, Luhrs DC, Van Grondelle R, Hegemann P, Kennis JTM: Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor. Proc Natl Acad Sci U S A 2006, 103:10895-10900. The photoactivation mechanism of BLUF domains was investigated by femtosecond transient absorption study with broadband white-light multichannel detection and extensive kinetic modeling. H/D exchange effects on the reaction dynamics revealed proton/hydrogen transfer steps in the photoreaction. A photocycle scheme involving anionic and neutral flavin radical intermediates was established and on basis of the BLUF crystal structure, a detailed molecular reaction mechanism was proposed. 14. Laan W, Gauden M, Yeremenko S, van Grondelle R, Kennis JTM, Hellingwerf KJ: On the mechanism of activation of the BLUF domain of AppA. Biochemistry 2006, 45:51-60. 15. Dragnea V, Waegele M, Balascuta S, Bauer C, Dragnea B: Timeresolved spectroscopic studies of the AppA blue-light receptor BLUF domain from Rhodobacter sphaeroides. Biochemistry 2005, 44:15978-15985. 16. Zirak P, Penzkofer A, Schiereis T, Hegemann PAJ, Schlichting I: Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides. Chem Phys 2005, 315:142-154. 17. Gauden M, Grinstead JS, Laan W, van Stokkum IHM, Avila Perez M, Toh KC, Boelens R, Kaptein R, van Grondelle R, Hellingwerf KJ, Kennis JTM: On the role of aromatic side chains in the photoactivation of BLUF domains. Biochemistry 2007, 46:7405-7415. Here, it is shown that electron transfer to FAD from a conserved tyrosine or a conserved tryptophan determines all photochemistry in BLUF domains. Electron transfer from tyrosine results in formation of the signaling state, while electron transfer from tryptophan provides a nonproductive deactivation pathway. 18. Grinstead JS, Hsu STD, Laan W, Bonvin AMJJ, Hellingwerf KJ, Boelens R, Kaptein R: The solution structure of the AppA BLUF domain: insight into the mechanism of light-induced signaling. Chembiochem 2006, 7:187-193. 19. Masuda S, Tomida Y, Ohta H, Takamiya K: The critical role of a hydrogen bond between Gln63 and Trp104 in the blue-light sensing BLUF domain that controls AppA activity. J Mol Biol 2007, 368:1223-1230. 20. Kennis JTM, Crosson S, Gauden M, van Stokkum IHM, Moffat K, van Grondelle R: Primary reactions of the LOV2 domain of phototropin, a plant blue-light photoreceptor. Biochemistry 2003, 42:3385-3392. 21. Schuttrigkeit TA, Kompa CK, Salomon M, Rudiger W, Michel-Beyerle ME: Primary photophysics of the FMN binding LOV2 domain of the plant blue light receptor phototropin of Avena sativa. Chem Phys 2003, 294:501-508. 22. Kennis JTM, van Stokkum IHM, Crosson S, Gauden M, Moffat K, van Grondelle R: The LOV2 domain of phototropin: a reversible photochromic switch. J Am Chem Soc 2004, 126:4512-4513. 23. Kotting C, Gerwert K: Proteins in action monitored by time-resolved FTIR spectroscopy. Chem Phys Chem 2005, 6:881-888. 24. Groot ML, Van Wilderen LJGW, Di Donato M: Femtosecond time resolved and dispersed infrared spectroscopy on proteins. Photochem Photobiol Sci 2007, 6:501-507. www.sciencedirect.com
This review gives a full account of femtosecond infrared spectroscopy technique along with applications to various photobiological systems. 25. Hellingwerf KJ, Hendriks J, Gensch T: Photoactive yellow protein, a new type of photoreceptor protein: will this ‘‘yellow lab’’ bring us where we want to go? J Phys Chem A 2003, 107:1082-1094. 26. van Stokkum IH, Larsen DS, van Grondelle R: Global and target analysis of time-resolved spectra. Biochim Biophys Acta 2004, 1657:82-104. 27. Ujj L, Devanathan S, Meyer TE, Cusanovich MA, Tollin G, Atkinson GH: New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy. Biophys J 1998, 75:406-412. 28. Changenet-Barret P, Espagne A, Charier S, Baudin JB, Jullien L, Plaza P, Hellingwerf KJ, Martin MM: Early molecular events in the photoactive yellow protein: role of the chromophore photophysics. Photochem Photobiol Sci 2004, 3:823-829. 29. Larsen DS, van Stokkum IHM, Vengris M, van der Horst MA, de Weerd FL, Hellingwerf KJ, van Grondelle R: Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump–dump–probe spectroscopy. Biophys J 2004, 87:1858-1872. 30. Groot ML, van Wilderen LJ, Larsen DS, van der Horst MA, van Stokkum IH, Hellingwerf KJ, van Grondelle R: Initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid-infrared spectroscopy. Biochemistry 2003, 42:10054-10059. 31. van Wilderen LJ, van der Horst MA, van Stokkum IH, Hellingwerf KJ, van Grondelle R, Groot ML: Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein. Proc Natl Acad Sci U S A 2006, 103:15050-15055. The isomerization of the p-coumaric acid chromophore in PYP was investigated by femtosecond infrared spectroscopy. Breaking of a hydrogen bond between the backbone and the chromophore was identified as a key step for successful entry into the photocycle. 32. Heyne K, Mohammed OF, Usman A, Dreyer J, Nibbering ETJ, Cusanovich MA: Structural evolution of the chromophore in the primary stages of trans/cis isomerization in photoactive yellow protein. J Am Chem Soc 2005, 127:18100-18106. The isomerization reaction of p-coumaric acid in PYP was characterized in great detail through femtosecond IR spectroscopy in combination with quantum-chemical calculations. 33. Groenhof G, Bouxin-Cademartory M, Hess B, De Visser SP, Berendsen HJC, Olivucci M, Mark AE, Robb MA: Photoactivation of the photoactive yellow protein: why photon absorption triggers a trans-to-cis lsomerization of the chromophore in the protein. J Am Chem Soc 2004, 126:4228-4233. 34. Premvardhan LL, van der Horst MA, Hellingwerf KJ, van Grondelle R: Stark spectroscopy on photoactive yellow protein, E46Q, and a nonisomerizing derivative, probes photoinduced charge motion. Biophys J 2003, 84:3226-3239. 35. Wagner JR, Brunzelle JS, Forest KT, Vierstra RD: A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome. Nature 2005, 438:325-331. 36. Yang X, Stojkovic EA, Kuk J, Moffat K: Crystal structure of the chromophore-binding domain of an unusual bacteriophytochrome RpBphP3 reveals residues that modulate photoconversion. Proc Natl Acad Sci U S A 2007, 104:12571-12576. 37. van Thor JJ, Ronayne KL, Towrie M: Formation of the early photoproduct Lumi-R of cyanobacterial phytochrome Cph1 observed by ultrafast mid-infrared spectroscopy. J Am Chem Soc 2007, 129:126-132. The structural dynamics of the open-chain tetrapyrrole chromophore of Cyanobacterial Phytochrome 1 on the picosecond timescale was revealed for the first time utilizing femtosecond infrared spectroscopy. Z/E isomerization of the C15 bond was found to occur corresponding to flipping of Ring IV. However, rather unexpectedly, significant torsional Current Opinion in Structural Biology 2007, 17:623–630
630 Biophysical methods
twisting around the C5 bond was observed at a previously considered structurally inactive part of the chromophore. 38. Kobayashi T, Saito T, Ohtani H: Real-time spectroscopy of transition states in bacteriorhodopsin during retinal isomerization. Nature 2001, 414:531-534. 39. Herbst J, Heyne K, Diller R: Femtosecond infrared spectroscopy of bacteriorhodopsin chromophore isomerization. Science 2002, 297:822-825. 40. Kukura P, McCamant DW, Mathies RA: Femtosecond stimulated raman spectroscopy. Annu Rev Phys Chem 2007, 58:461-488. This review provides a comprehensive overview of femtosecond-stimulated Raman spectroscopy and its application to various photoactive systems. 41. Kukura P, McCamant DW, Yoon S, Wandschneider DB, Mathies RA: Structural observation of the primary isomerization in vision with femtosecond-stimulated Raman. Science 2005, 310:1006-1009. The isomerization mechanism of rhodopsin was revealed in unprecedented detail with femtosecond-stimulated Raman spectroscopy. Isomerization around the C11 C12 double bond was found to occur in less than 200 fs. Further evolution to a strained all-trans structure took place in 1 ps. 42. Schoenlein RW, Peteanu LA, Mathies RA, Shank CV: The 1st step in vision—femtosecond isomerization of Rhodopsin. Science 1991, 254:412-415.
Current Opinion in Structural Biology 2007, 17:623–630
43. McCamant DW, Kukura P, Mathies RA: Femtosecond stimulated Raman study of excited-state evolution in bacteriorhodopsin. J Phys Chem B 2005, 109:10449-10457. This paper concerns a femtosecond-stimulated Raman study on bacteriorhodopsin. Although no dynamic structural information is obtained during the first picosecond because of distortion of the signals by ground-state Raman features, evidence is provided for dissipation of Franck-Condon active modes of retinal into reactive low-frequency modes. 44. Lenz MO, Huber R, Schmidt B, Gilch P, Kalmbach R, Engelhard M, Wachtveitl J: First steps of retinal photoisomerization in proteorhodopsin. Biophys J 2006, 91:255-262. Here, a first full account of the excited-state reactions of retinal in proteorhodopsin is given as probed with visible ultrafast spectroscopy. Isomerization of all-trans retinal was found to proceed in a way similar to that of bacteriorhodopsin. 45. Pollard WT, Mathies RA: Analysis of femtosecond dynamic absorption spectra of nonstationary states. Annu Rev Phys Chem 1992, 43:497-523. 46. Kahan A, Nahmias O, Friedman N, Sheves M, Ruhman S: Following photoinduced dynamics in bacteriorhodopsin with 7-fs impulsive vibrational spectroscopy. J Am Chem Soc 2007, 129:537-546. This paper concerns a comprehensive study of impulsive vibrational spectroscopy (IVS) on bacteriorhodopsin. It is demonstrated that the IVS signals mainly arise from an impulsive stimulated Raman process creating wavepackets on the ground-state potential energy surface, and thus limited information on excited-state dynamics is obtained.
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