Phase Transitions Vol. 77, Nos. 1–2, January–February 2004, pp. 3–20
MEASURING AND MODELLING DYNAMICAL CHANGES IN THE STRUCTURE OF PHOTOACTIVE YELLOW PROTEIN JOCELYNE VREEDE, MICHAEL A. VAN DER HORST, REMCO KORT, WIM CRIELAARD and KLAAS J. HELLINGWERF* Microbiology Group, Swammerdam Institute for Life Science, University of Amsterdam, Nieuwe Achtergracht 166, NL-1018 WV Amsterdam, The Netherlands Biological photoreceptors are very suitable for studies of the structure–function relationship in proteins. Of the many possible candidates, from at least six different families, we discuss here photoactive yellow protein (PYP, a member of the Xanthopsin family). The excellent physical and photochemical stability of PYP has allowed recording of a large amount of data relevant for this characteristic. The timescales relevant for functional transitions in PYP are most easily studied with transient spectroscopy. Application of the combination of UV/Vis and FTIR spectroscopy has revealed (besides multiple electronically excited states) the subsequent involvement of the following transient intermediates: I0, pR1, pR2, pB0 , pB and pBdeprot. The structures of these transient intermediates have been characterized with general structural techniques like X-ray diffraction and NMR spectroscopy. Most extensive results have been obtained with X-ray diffraction, applied both in a time-resolved mode and by making use of low-temperature trapping. This has led to a detailed description of the reversible spatial changes in PYP that follow light activation. The structure of the transient intermediates, however, is exquisitely sensitive to constraints from the molecular environment of the protein, like the absence/presence of a crystalline lattice. Extrapolating these results to the signalling state of PYP in vivo is extremely complicated but also very challenging. Rather than measure, one can also simulate and/or calculate the spatial structures of transient intermediates of PYP. This challenge is most straightforwardly tackled with a detailed analysis of the intrinsic dynamics of the stable receptor state of PYP. Such studies have revealed striking similarities in the dynamics of all PAS domain proteins of which a spatial structure has been determined. The next challenge is to properly describe the change in configuration of the chromophore, induced by light, and to expand such simulations to the timescale relevant for completion of this photocycle. Keywords: Photoactive yellow protein (PYP); p-Coumaric acid; Low-temperature trapping; Time-resolved X-ray diffraction; Molecular dynamics; Photocycle; Transient intermediate
1. PHOTORECEPTOR FAMILIES Light-sensing proteins (or photoreceptor proteins) are optimally suited to study the role of dynamical alterations in protein structure in relation to their biological function. Such proteins can be triggered with ultra-short light pulses, and therefore excellent
*Corresponding author. Tel.: þ31 20 5257055. Fax: þ31 20 5257056. E-mail:
[email protected] ISSN 0141-1594 print: ISSN 1029-0338 online ß 2004 Taylor & Francis Ltd DOI: 10.1080/01411590310001621384
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time-resolution is achievable in studies of the dynamical alterations in their structure. Additionally, because these photoreceptor proteins are signal-transduction proteins, one may anticipate large conformational transitions to be involved in their signalling state formation (and subsequent decay). Furthermore, the (changing) colour of these proteins is an excellent indicator for the timescales relevant to resolve structural transitions. Largely because of this, detailed insight along such lines has been obtained for a number of different photoreceptor proteins. Hence they are excellent model systems to understand atomic detail of the configurational and conformational transitions, between receptor and signalling states, required for photoreceptor functioning. The many different photoreceptor proteins that have been described in the literature can be classified into a limited number of families. The most rational approach to do this is to base this classification on the chemical structure of their light-absorbing chromophores. Following this classification, the most important families are the rhodopsins (Oesterhelt, 1998; Pepe, 1999), the phytochromes (Quail, 1998) and the xanthopsins (Kort et al., 1996). Strikingly, in all three families E/Z isomerization of the light-sensitive chromophore (a retinaldehyde, a linear tetrapyrrole and a p-coumaric acid, respectively) is the photochemical basis of their activation. During the past few years, however, several additional photoreceptor proteins have been discovered, that all happen to bind a flavin derivative. For these latter, therefore, additional arguments (derived from protein sequence alignment) are used for further classification. Accordingly, the families of the cryptochromes (Ahmad and Cashmore, 1993), the phototropins (Briggs and Christie, 2002) and the BLUF proteins (Gomelsky and Klug, 2002) have been proposed. It will be evident that the primary photochemistry of these latter photoreceptor proteins cannot be based upon E/Z isomerization. New and intriguing types of primary photochemistry have been uncovered in these latter families instead, like transient cysteinyl-adduct formation in the LOV domains of phototropins (Crosson and Moffat, 2002), whereas BLUF proteins may be activated by light-induced proton transfer (Laan et al., 2003) and the activation of cryptochromes may be based on light-induced electron transfer (Galland, unpublished observations). Nevertheless, in all photoreceptor proteins the light-induced change in configuration of the chromophore subsequently leads to formation of a signalling state of sufficient stability and altered conformation so that the process of photon absorption can be communicated to a downstream signal transduction partner.
2. THE PROTOTYPE XANTHOPSINS: PHOTOACTIVE YELLOW PROTEIN Members of the xanthopsin family contain a coumaryl derivative as their light-sensitive chromophore. The prototype member of this family, photoactive yellow protein (PYP; for recent reviews see Cusanovich and Meyer (2003), Hellingwerf and Gensch (2003)) was discovered in 1985 in the purple-sulfur bacterium Ectothiorhodospira halophila. In this organism (currently referred to as Halorhodospira halophila) it functions as a blue-light repellent photoreceptor (Sprenger et al., 1993). PYP is a relatively small (14 kDa; 125 amino acids) and highly water-soluble photosensor protein. This protein is very stable, physicochemically as well as photochemically, presumably because its chromophore contains only a single C¼C bond that can isomerize. Furthermore, apo-PYP is easily over-produced heterologously in Escherichia coli, after which holo-protein can be reconstituted via the use of imidazole esters of 4-hydroxy-cinnamic
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FIGURE 1 In vitro reconstitution of apo-PYP. First, activated imidazole esters of 4-hydroxy-cinnamic acid are formed. The activated ester then reacts spontaneously with the single cysteine (Cys 69) of PYP.
acid (see Fig. 1). Accordingly, the holo-protein can be produced and purified in large quantities, after which it readily crystallizes into well-diffracting crystals. For these reasons PYP has become a prototype system for experimental and/or computational studies of the spatial structure of both stable and of transient intermediate states of photoreceptor proteins, see Genick et al. (1997, 1998); Perman et al. (1998); Ren et al. (2001). The structure of the stable ground state of PYP has been determined initially via X-ray crystallography (Borgstahl et al., 1995). The results revealed that it has an /-fold, containing a six-stranded anti-parallel -sheet as a scaffold, flanked by several helices. It contains two hydrophobic cores, one on each side of the -scaffold. The smaller of these two comprises the N-terminus, while the larger contains the chromophore-binding pocket. This p-coumaric acid chromophore is linked through a thiol–ester linkage to the sole cysteine in the protein, Cys69. In the ground (or receptor) state this chromophore resides in the trans configuration and is deprotonated. The resulting negative charge is stabilized via a hydrogen-bonding network, involving the residues Y42, E46, and T50. Additional stabilization may be provided by the positive charge on R52 (Groenhof et al., 2002). Besides a crystal structure, a solution structure has also been determined, using multinuclear NMR analyses (Dux et al., 1998). Though minor differences exist, in particular in the region of the N-terminal helices of the protein, molecular dynamics simulations have shown that both structures are essentially the same (Antes et al., 2002).
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3. MEASURING FUNCTIONAL ACTIVITY OF PYP IN VITRO: THE PHOTOCYCLE The key in vitro functional characteristic of PYP is that it goes – after absorption of a blue photon – through a reversible photocycle. This photocycle (see Fig. 2) can be divided into three basic steps: (a) Initial photocycle events, in which the chromophore isomerizes. These initial events are considered completed once the most stable red-shifted intermediate (pR) is formed; (b) Protonation of the cinnamyl chromophore through intra-molecular proton transfer and subsequent structural change of the protein, which under many circumstances is so extensive that it is best characterized as a partial unfolding of the protein (see further below). In this step the signalling state, pB, is formed. As we shall see later, the extent of structural change in this step depends on the mesoscopic (¼ molecular) environment in which the protein resides; (c) De-protonation and re-isomerization of the chromophore, and refolding of the protein. In these steps the ground state, pG, recovers. It should be noted that over the years three different nomenclatures for this photocycle have evolved: the alternatives for the nomenclature used here (i.e. pG, pR, and pB, introduced in Hoff et al. (1994b) are P, I1 and I2 (Meyer et al., 1987) and PYP, PYPL, and PYPM (Imamoto et al., 1996)). The key aspect of the molecular function of PYP that needs to be resolved is the question: ‘‘How is the very local change in configuration of the p-coumaryl chromophore (induced by light absorption) translated into a change in conformation on the surface of the protein, that can be detected by, and transmitted through, the downstream signal transduction chain that communicates the absorption of a blue photon to the flagellum of the bacterium?’’ (see Fig. 3). In the following we will discuss various aspects of the answer(s) to this question. First of all it is important to have an estimate of the timescale at which the underlying partial reactions, which lead to formation of the relevant intermediate states involved, take place. This is most easily studied with (femtosecond) time-resolved
FIGURE 2 Basic PYP photocycle scheme. Only the key intermediates and basic photocycle events are shown. Three basic steps can be distinguished: (i) The light-driven isomerization of the chromophore, (ii) the formation of the signalling state pB, and (iii) the recovery of the ground or receptor state.
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FIGURE 3 General scheme describing (photo)taxis responses in Bacteria and Archaea, based upon the archaetype system of chemotaxis in Escherichia coli. Photoreceptors like PYP may interact with MCP-like proteins to modulate kinase activity of a CheA-like histidine proteine kinase. The arrows with a question mark indicate that also still unknown pathways of signal input into Che-like chemosensory systems exist. MCP: methyl-accepting chemotaxis protein.
optical spectroscopy. Multiple transient absorption- and fluorescence spectroscopy studies have aimed to answer this question; they resulted, however, in partially conflicting results. The most recent consensus sequence of intermediates, representing the initial phase of the photocycle of PYP in which the trans to cis isomerization of the chromophore is effectuated, is as follows: pG ! ðpG Þ2 ! ½ I0 ! pR1 ð¼ I1 Þ ! pR2 :
ð1Þ
This scheme indicates that a heterogeneous electronically excited state, (composed of (at least) two separate states), formed through the absorption of a blue photon, relaxes to an initial intermediate (with a lifetime of a few hundred picoseconds (I0 )). Some z studies have postulated the existence of an additional intermediate (I0 ), before the stable, well-characterized nanosecond red-shifted intermediate pR1 (or: I1 ) is formed, but more recent results ascribe the measured phenomena that led to the proposal z of the existence of I0 to a very slow decay of part of the stimulated emission from pG (Larsen et al., submitted for publication). The transition from pR1 to pR2 is accompanied by only very small changes in the colour of the protein; this transition was only clearly detected upon application of photo-acoustic spectroscopy. For review, see Hellingwerf and Gensch (2003). All these ultra-fast transient ground-state intermediates are more (I0 ) or less (pR) red-shifted as compared to the colour of the stable ground state, consistent with the interpretation that the structural transitions in these transient states are largely limited to the isomerization process of the chromophore. Nevertheless, these spectral studies have revealed additional structural detail about these intermediates: The displacement of the C7, 8 carbon atoms of the chromophore, necessary for the actual isomerization to take place, occurs very early in the reaction sequence. Initially this was not directly obvious because it was generally assumed
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that this process would only be completed after full relaxation of the excited state(s), which has some (very) slow components (Changenet et al., 1998). Time-resolved femtosecond UV/Vis polarization spectroscopy indicated – through the measured changes in anisotropy – that already in I0 isomerization must have taken place (Gensch et al., 2002). Very recently, femtosecond time-resolved FTIR measurements showed that isomerization is among the very first processes detectable (i.e. within a few hundred femtoseconds, (Groot et al., 2003)). The same FTIR technique, applied on a low-temperature trapped pR intermediate, had already revealed that in that state the hydrogen bond between E46 and the chromophore was still intact (Xie et al., 1996), implying that a multiple-bond isomerization process (in which the phenolate part of the chromophore hardly moves) must be the structural basis of photoactivation of PYP. The femtosecond FTIR studies also indicate that the I0 to pR1 transition is accompanied by structural transitions in the tail part of the chromophore, transitions that appear to be significantly restricted at low temperature. The transition from pR1 to pR2 is most probably due to rearrangements of side chains of the protein that surround the chromophore. The slow phase of the photocycle is accompanied by much more dramatic changes. First, the colour of the PYP protein changes dramatically from yellow (in the pG state) to almost colourless in pB, caused by intra-molecular proton transfer from E46 to the chromophore, and subsequently the protein transiently, and reversibly, partially unfolds. Before recovery to the receptor state (or the stable ground state) the chromophore must first again be deprotonated to the phenolate form: pR2 , pB0 ! pBð I2 Þ , pBdeprot ! pG:
ð2Þ
A further complication is hidden in the fact that the mechanism of initiation of the photocycle of PYP, by absorption of a blue photon, appears to be temperature dependent (Imamoto et al., 1996): At low (e.g. at 77 K) temperature two intermediates (PYPB and PYPH) are formed in parallel that – after transition through two additional intermediates, PYPHL and PYPBL – converge in an intermediate that is very similar to pR (or: I1). The PYPB=BL intermediates show most similarity to the I0 intermediate.
4. CRYSTALLOGRAPHIC STUDIES ON PYP PHOTOCYCLE INTERMEDIATES Structural changes that are associated with the formation and decay of optical intermediates have been analyzed in detail by a wide range of techniques, including X-ray crystallography (Genick et al., 1997, 1998; Perman et al., 1998; Ren et al., 2001), FTIR-spectroscopy (Brudler et al., 2001; Imamoto et al., 2001; Xie et al., 2001), and NMR-spectroscopy (Rubinstenn et al., 1998). The application of these techniques often demands widely different sample conditions; hence these methods yield not only different, but also often complementary information about the structure and kinetics of the intermediates involved. For example, for the application of X-ray diffraction analysis, which is further discussed below, the protein obviously needs to be in a crystalline state. This state, however, may put constraints on the allowable structural changes, and their kinetics, because of the numerous intermolecular crystal
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lattice contacts and the relatively low water content in the crystal. Also the use of highly concentrated protein samples, necessary to apply the latter two techniques, may affect the kinetics and even the pathway of formation of structural intermediates. The effect(s) of specific sample conditions on the kinetics of photocycle transitions are most conveniently investigated by using transient absorption spectroscopy, as the formation of specific photocycle intermediates has initially been defined via the associated changes in the absorption spectrum of the protein (see above). Absorption spectroscopy does not only allow monitoring of the presence of the optical intermediate of interest and/or of additional intermediates, but also provides a tool to find the experimental conditions to obtain the highest occupancy for a particular intermediate state. In addition, relevant reaction kinetics can be determined under the particular sample conditions needed for the various techniques, such as a thin protein film or a crystal, possibly at a reduced hydration level, or an extreme salt concentration and/or temperature. Possible constraints on conformational changes, for example due to low water content or intermolecular contacts in the crystal lattice, can thus be identified. Absorption measurements on PYP crystals turn out to be much more complex than those on PYP in solution, because the small dimensions and extremely high optical density in the blue region of the spectrum of these crystals demand a very stable and well-focussed monitoring light beam. In the three absorption studies on PYP crystals described to date, a micro-spectrophotometer was used to identify the optically distinct photocycle intermediates (Ng et al., 1995; Kort et al., 2003; Mano et al., 2003). These optical studies on PYP crystals have revealed that the kinetic model for PYP in solution does not entirely match that for crystalline PYP, as described in more detail below.
5. MONITORING STRUCTURES OF EARLY PHOTOCYCLE INTERMEDIATES Two approaches can be used to monitor structural changes associated with the formation of early intermediates by X-ray crystallography. Early intermediates are defined here as those formed in the picoseconds to slow nanoseconds time range. By far the most common method to elucidate structures of early intermediates is cryo-trapping, in which low temperature, obtained with liquid ethane or liquid nitrogen, stops further thermal relaxation of a light-excited protein crystal. Besides cryotrapping, which is further discussed below, a second and more complicated approach is to use extremely short X-ray pulses of high intensities to obtain a snap shot of PYP while it progresses through the photocycle, in the so-called time-resolved Laue diffraction approach (Wulff et al., 1997; Moffat 2001). By this method the structure of a nanosecond photocycle intermediate has been described that shows that the isomerization reaction of the ethylene bond of the chromophore is accompanied by minimal structural rearrangements in the protein (Perman et al., 1998; Ren et al., 2001). The aromatic ring of the chromophore remains fixed in the same plane, while the thiol–ester bond to Cys69 flips in a crankshaft motion to account for the photoisomerization reaction. The advantage of the use of time-resolved X-ray crystallography over cryo-trapping techniques is that low-temperature artefacts, due to limited protein flexibility in frozen crystals, are eliminated. However, other factors, such as the inefficiency of bleaching crystals in stroboscopic experiments and radiation
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damage by light or X-rays at non-cryogenic temperatures, contribute to the difficulty of applying this Laue technique. Despite these difficulties, time-resolved X-ray crystallographic studies eventually led to a four-dimensional model for the lightinduced photo-isomerization reaction and subsequent conformational changes (Ren et al., 2001; Schmidt et al., 2003). The observed early structural rearrangements in the protein include chromophore isomerization and a transient flip of the C9 carbonyl group, while the H-bond of the phenolate moiety of the chromophore to Glu46 remains intact. These observations are in line with other independent studies, for example with FTIR spectroscopy (Xie et al., 1996, Groot et al., 2003), indicating the feasibility of time-resolved X-ray crystallography to elucidate transient protein structures in a crystal (Ren et al., 2001). Flash-freezing a crystal can also lead to trapping of early photocycle intermediates. This method requires a very rapid freezing procedure for which usually liquid ethane is used, for reasons of thermal conductivity. In addition, it requires synchronization of a light flash with the freezing procedure. Very elegant methods have been developed to tackle the latter problem in studies with other photoreceptor proteins, such as freezing two-dimensional crystals of bacteriorhodopsin in liquid ethane at varying times after illumination with a light flash (Subramaniam et al., 1999). However, only milliseconds time resolution is accessible with this approach. For PYP this approach has not been applied so far. An alternative way of cryo-trapping early intermediates is the continuous illumination of a protein crystal at low temperatures, although strictly speaking this is not cryo-trapping, because a photostationary state of intermediates will be formed. The exposure of a PYP crystal to blue-light of 400 40 nm at 100 K leads to the formation of such a photostationary state of intermediates, which includes at least the bathochromic intermediate PYPB and the hypsochromic intermediate PYPH (Fig. 4A). No significant differences have been observed between the low-temperature photochemistry in PYP crystals and in frozen glycerol solution (Hoff et al., 1992; Imamoto et al., 1996; Kort et al., 2003). Apparently, the light-induced formation of photoproducts at very low temperatures is not affected by the crystal lattice. Comparison of the low-temperature studies on crystalline PYP described here with those carried out on the photo-isomerizable photoreceptor protein bacteriorhodopsin (BR) under similar conditions led to the identification of a number of striking similarities and differences. The K intermediate of BR can be considered to be the equivalent of PYPB, as this is the first photoproduct of the photocycle of BR that can be stabilized at liquid nitrogen temperatures and its formation is accompanied by a 40-nm red-shift (from 578 to 608 nm). In contrast to PYPB, K is formed with a high-quantum yield even at 4 K and is stable in the dark up to 130 K (Balashov and Ebrey, 2001). It is obvious from the absorption spectra in Fig. 4A that only a relatively small fraction of the dark state of PYP can be converted to PYPB under the experimental conditions used. A maximal occupancy of approximately 20% for PYPB can be calculated from the relative depletion of the absorption band originating from the ground state of PYP, while at least 50% of BR can be converted to K at 100 K (Xie, 1990). Possibly, the much lower efficiency of the PYP to PYPB conversion results from the simultaneous formation of the blue-shifted intermediate PYPH, a side reaction that has not been observed in the BR low-temperature photocycle, or to differences in the relevant quantum yields. Another striking difference is the instability of PYPB even at 100 K, as shown in the inset in Fig. 4B. The majority of PYP in the red-shifted PYPB state decays in the dark at 100 K with a time constant of approximately 100 s,
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FIGURE 4 Blue-light-induced absorption changes in a single P65 PYP crystal at 100 K. (A) The solid line indicates the reference absorption spectrum of a single P65 crystal at 100 K. The dashed line refers to the absorption spectrum of the same crystal illuminated with blue-light of 400 40 nm for a period of 60 s at 100 K. The inset shows the difference spectrum between the absorption spectrum of the blue-light illuminated crystal and the reference absorption spectrum. Spectra were monitored with the continuous Xenon light source (reproduced from Photochemistry & Photobiology, with permission from the editor). (B) Effect of thermal relaxation in the dark on the occupancy of PYPB in P65 PYP crystals. Crystals were illuminated with blue-light of 400 40 nm for a period of 60 s at 100 K. Time interval of spontaneous PYPB relaxation was varied in the absence of actinic light. Spectra were obtained by averaging 10 traces monitored with a 1.3 ms FWHM-pulse of a Xenon flash lamp (adapted with permission from Photochemistry & Photobiology).
while part of it is trapped for a much longer period. An increase in the temperature, e.g. achievable by blocking the nitrogen cryostream, led to the disappearance of the residual absorption band at 490 nm. This complicated behaviour of PYPB at low temperature may indicate the presence of a mixture of low-temperature species with similar, red-shifted absorption spectra. However, further decrease to liquid helium temperatures (4–9 K) did not resolve this point, as in contrast to the situation in BR
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(Iwasa et al., 1979), in PYP photochemical reactions were not observed at those temperatures (Ravelli et al., unpublished data; van der Horst et al., unpublished data). The stability of low-temperature intermediates is essential for the structural characterization of these intermediates by X-ray diffraction studies that require relatively long exposure times towards X-rays. One way to overcome the decrease in occupancy of intermediates resulting from thermal decay is the use of continuous actinic illumination during data collection, as has been applied for elucidation of the structure of the low-temperature intermediate, tentatively assigned to PYPBL (Genick et al., 1998). However, it is relevant to note that the trapped intermediates of photoactive proteins are photoactive themselves. Thus, continuous illumination of these crystals increases the risk of formation of secondary photoproducts.
6. MONITORING THE STRUCTURE OF A LATE PHOTOCYCLE INTERMEDIATE At room temperature pB, in crystalline PYP, decays approximately 15 times faster than pB in solution (Ng et al., 1995), where this time constant has been determined to be 150 ms (Meyer et al., 1987; Hoff et al., 1994b). Formation and decay of pB in crystals is therefore much more easily observed under conditions where pG recovery is slowed down by decreasing the temperature to 12 C (Genick et al., 1997). This has been confirmed at the level of absorption changes by Ng et al. (1995) and in recent spectroscopic studies on PYP crystals by Kort et al. (2003). Both studies show that under these conditions decay of pB is well described with a bi-exponential function with time constants of ¼ 0.78 s (explaining 70% of the amplitude) and ¼ 0.25 s (30%; Fig. 5). These results may indicate the occurrence of parallel pathways in the photocycle of photoactive yellow protein, as also indicated in the photocycle schemes by Hoff et al. (1994a) (for the pR to pB transition) and Ng et al. (1995) for pB recovery. It is a challenge to resolve the structural basis of these parallel processes. The fast pB decay kinetics in crystalline PYP suggests that in a crystal the protein goes through a short-cut version of the photocycle. FTIR studies indicate that lightinduced structural changes in the protein backbone associated with pG recovery are dramatically reduced in PYP crystals (Xie et al., 2001). In addition, the point was raised that partial dehydration of the protein in a crystal is probably contributing to the suppression of large structural changes upon pB formation. This is in agreement with the findings in this work and with recent observations indicating fast pB decay kinetics in partially dehydrated PYP films (van der Horst et al., unpublished data). In this latter study it was shown that moderately reduced humidity affects the photocycle kinetics. Most notably, ground state recovery was accelerated, whereas the rate of pR depletion was decreased. Because the rates of the latter two processes were rather similar, this suggests that under these conditions an alternative (short-cut) pathway to ground state recovery is operating, possibly without involvement of the pB state(s). Additionally, conformational changes in PYP in such films may not occur to the same extent as in solution, because they could be limited by intermolecular contacts, just as in a crystal lattice. This is in line with the concept of photoactive yellow protein being a photoreceptor protein with a rigid core surrounded by a soft body, in which small light-induced changes in the chromophore configuration lead to large
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FIGURE 5 Kinetic analysis of pB relaxation in a single crystal at 12 C. A P63 crystal was mounted in a cryoloop after soaking in paraffin oil. It was illuminated with the 488-nm laser line of a CW Argon ion laser for a period of 1 s. The optical density at 350 nm has been plotted against time. A bi-exponential fit is represented as the full line from 2 to 8 s. The residuals show the increase in the quality of the bi-exponential over the mono-exponential fit (figure copied from Photochemistry & Photobiology, with permission from the editor).
changes in protein conformation (Reat et al., 1998). The molecular environment of the protein easily affects these latter changes, and the rate at which they occur, in contrast to changes in the rigid core. Considering this model it is surprising that Mano et al. (2003) report that primary photochemistry of PYP is affected by the degree of its crystallinity. The results shown in Figs. 4 and 5 suggest that the low-temperature photocycle, and thus probably also early photocycle intermediates, involving relatively small amplitude structural changes, can be accurately resolved by crystallographic techniques. The altered photocycle kinetics in crystals – as compared to PYP in solution – indicates that for the description of conformational changes of larger amplitude, on the milliseconds to seconds timescale, besides X-ray diffraction data, independent evidence obtained with complementary techniques, such as FTIR spectroscopy and NMR spectroscopy, is important. Recently, we extended our work at low temperature via the analysis of the structure of a PYP intermediate trapped at temperatures of 100 K and below. This choice of temperature range makes it likely that we have trapped a very early intermediate of PYP. In contrast to Genick et al. (1998) we do observe complete trans to cis isomerization of the coumaryl chromophore upon illumination of the protein with blue light under these conditions (Ravelli et al., in preparation).
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7. DYNAMICS OF PYP IN SILICO As was already discussed above, several detailed studies, using Laue diffraction and cryo-crystallography (Genick et al., 1997; Genick et al., 1998; Perman 1998), NMR spectroscopy (Rubinstenn et al., 1998), small-angle X-ray scattering (Imamoto et al., 2002, Sasaki et al., 2002), transient UV/Vis- (Van Brederode et al., 1996) and FTIR spectroscopy (Hoff et al., 1999; Xie et al., 2001) have all revealed that during the photocycle of the photoactive yellow protein distinct and significant configurational and conformational changes do occur. It is these conformational changes that are generally believed to be the biological signal that is translated into a cellular response, via subsequent protein–protein interactions. To correlate these data with the available high-resolution structure of PYP and to study possible protein motions involved in the photocycle, PYP structural dynamics have been investigated by computer simulation (van Aalten et al., 1998). First, molecular dynamics simulations were performed with the aim of identifying concerted backbone motions in PYP. These simulations were subsequently analyzed using so-called essential dynamics (Amadei et al., 1993). Application of the essential dynamics method on the results of the molecular dynamics simulations of PYP (van Aalten et al., 1998) revealed large, chromophorelinked fluctuations of the protein in the ground state, in particular of loops that are part of the core PAS domain motive of PYP. This PAS domain is the prototype of a structural module that can be found in proteins in all kingdoms of life (Pellequer et al., 1998; Taylor and Zhulin, 1999). The PAS (PER–ARNT–SIM) module was first identified in the Drosophila clock protein PER and the basic-helix-loop-helixcontaining transcription factors aryl hydrocarbon receptor nuclear translocator (ARNT) in mammals and single-minded protein (SIM) in insects (Nambu et al., 1991). Most PAS domains are sensory modules, typically sensing oxygen tension, redox potential or light intensity and/or colour (Nambu et al., 1991; Zhulin et al., 1997; Taylor and Zhulin, 1999); alternatively they mediate protein–protein interactions and/or register the presence of small ligands (Anantharaman et al., 2001). Although the amino acid sequence of the different PAS domains shows little similarity, their three-dimensional fold appears to be significantly conserved. The structure of all PAS domains resembles the structure of photoactive yellow protein (Pellequer et al., 1998). The molecular dynamics simulations and essential dynamics analyses (later supported by X-ray crystallographic studies (van Aalten et al., 2000)) pointed to distinct conserved glycines that were functioning as hinge points, which allow mutual flexibility to sub-structures in the protein. Figure 6 shows this conserved hinge-bending function around conserved glycines in an alignment of five different PYP proteins. These glycines, in particular Gly 47 and Gly 51, all fall within the PAS fold. The conserved glycines and prolines in the PAS domain are assumed to be important for the structure and/or conformational flexibility in the entire PAS domain (van Aalten et al. 1998). Furthermore, it was suggested that PAS domain dynamics is a conserved property in these signal transduction modules, and that their biological function is performed via these conserved intrinsic functional dynamical properties. This hypothesis was substantiated in a recent study on a series of PAS-domaincontaining proteins of which a high-resolution X-ray structure is available, including PYP (Vreede et al., 2003). The use of CONCOORD (de Groot et al., 1997), followed by the application of essential dynamics analyses, indeed revealed highly similar concerted motions in the different PAS domains. Furthermore, these similar motions
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FIGURE 6 Sequence alignment of PYP isolated from various species. Ehal ¼ Ectothiorhodospira halophila, Csal ¼ Chromatium salexigens, Rsph ¼ Rhodobacter sphaeroides, Rsal ¼ Rhodothalassium salexigens, Rcap ¼ Rhodobacter capsulatus. Conserved residues are indicated in grey boxes. Grey scale in the bar at the bottom of the alignment indicates degree of hinge-bending character for the indicated residues; white: no hingebending, dark grey: strongest hinge-bending character.
were indeed conserved concerted motions, supporting the hypothesis that the common structure of PAS domains implies common flexibility and that it is this conserved property that is fundamental for PAS domain function in signal transduction. Translated back to protein structure, the motions along the first three modes of conformational flexibility of PYP are compared in Fig. 7. The central -sheet appears to be relatively static, whereas the loops, most notably the / segment, show the largest fluctuations. In the PAS domain, this segment is generally considered important for the binding of a ligand. A recent analysis of LOV domains has revealed that the / region participates in a conserved salt bridge. Breakage of this bridge is an early step towards signalling state formation. It has been proposed that this step is crucial for signal transduction (Crosson et al., 2003). In a follow-up study the influence of intermolecular contacts of PYP in a crystalline lattice on the structure of its transient states is being investigated with the molecular dynamics simulation technique. In the crystalline state PYP proceeds through a photocycle, although X-ray diffraction studies of cryo-trapped photocycle intermediates show only little associated conformational change in backbone structure (Genick et al., 1997). Earlier work on the comparison of egg white lysozyme in an aqueous buffer and in a crystalline lattice, using computer simulation, indicates that protein conformation is not significantly altered by the intermolecular interactions present in
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FIGURE 7 PYP conformational changes. Atomic positional shifts as described by the first three eigenvectors of PYP are depicted as structures in Cartesian space. Two structures are depicted corresponding to 2 nm (coloured) and þ 2 nm (transparent) along the eigenvectors. Relative degrees of positional shifts are indicated from blue (smallest fluctuations) to red (largest fluctuations). The transparent structures indicate the direction of the motion.
a crystal lattice (Stocker et al., 2000), but that the amplitude of fluctuations of flexible loops is larger in solution. This hampering of the conformational freedom of functional flexible loops of PYP by the constraints imposed by the crystalline lattice in MD simulations is currently being characterized via comparison with the dynamics and flexibility of PYP in aqueous solution (Vreede et al., unpublished). The conformational modes described above eventually lead to the transduction of a signal, initiated by the signal itself (i.e. a blue-light photon for PYP). This is the trigger that leads to conformational changes that mediate signal transduction. These initial structural and conformational changes have been investigated with computer simulation techniques. The first attempt to simulate photon absorption, via the change in (electron) charge distribution upon excitation, already pointed to the significance of the surrounding amino acids in the binding pocket (Yamato et al., 1998). Nevertheless, the measured change of the transition dipole moment in PYP is much larger (Premvardhan et al., 2003). Yoda and co-workers have employed an approach derived from first principles to further elucidate the importance of the amino acid residues that surround the chromophore, especially those contributing to the hydrogen-bonding network of its phenolate moiety (Yoda et al., 2001). It is this specific environment that allows PYP to sense blue light, since the stabilization of the negative charge on the chromophore by the hydrogenbonding network decreases the pKa of the phenolic moiety of the chromophore, so that it is anionic at neutral pH and due to that more red-shifted in its spectral properties than its protonated counterpart. The density functional theory (DFT) approach corroborates the importance of the hydrogen-bonding network for the spectral tuning of the protein. Furthermore, DFT calculations show that the initial event of isomerization is not the destabilizing factor that leads to partial unfolding of the protein. Sergi et al. (2001) show that the protein environment lowers the rotational barrier for trans to cis isomerization. The chromophore in the cis configuration also fits into the binding pocket (Sergi et al., 2001), but alters the conformation of the protein to facilitate subsequent intramolecular proton transfer.
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It is the intramolecular proton transfer, accompanied by the simultaneous re-distribution of charge on the chromophore, that causes the disruption of the hydrogen-bonding network, thus destroying the intramolecular interactions that stabilize the folded state of the protein (Groenhof et al., 2002). Proton transfer, however, can only occur under optimal conditions in the binding pocket, i.e. only for specific combinations of side-chain orientation, proton transfer from Glu46 to the chromophore is energetically favourable (Antes et al., 2002). The actual unfolding of PYP in the metastable pB state has been observed in silico as well. Shiozawa et al. (2001) performed a molecular dynamics simulation starting from the crystal structure of a photobleached intermediate. Starting in this transient intermediate state, the hydrogen bond between Arg 52 and the phenolic oxygen of the chromophore is being disrupted, thus allowing water molecules to enter the chromophore-binding pocket. The subsequent invasion of water molecules into the protein causes a rearrangement of the hydrogen-bonding network, facilitated by large fluctuations of the protein backbone surrounding the binding cavity. As a result the embedded residue Glu46 becomes hydrated (Shiozawa et al., 2001). These results are indicative of the wide gap that still exists between accepting the trigger that leads to signal transduction (i.e. light absorption) and the actual signal transduction itself. The initial stages of the photocycle require interactions to be described at the level of quantum theory, whereas the large alterations of protein conformation, required for signal transduction, are sufficiently described by simple classical mechanics, based on empirical observations. Regarding the (quantum) mechanical modelling of the movement of atoms in the initial phase of the response of PYP, related calculations on retinal-containing photoreceptor proteins may lead the way, see e.g. Buss et al. (1998), Gonzalez-Luque et al. (2000).
Acknowledgement This work was supported by the Dutch Science Research Council (NWO).
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