Rhodopsin(s) in eubacteria

While the biochemical basis of photosynthesis by bacteriochlorophyll-based reaction centres in purple phototrophic Eubacteria and retinal-based bacteriorhodopsin in the Archaebacterium Halobacterium salinarium has been elucidated in great detail, much less is known about photosensory signal transduction; this is especially the case for Eubacteria. Recent findings on two different photosensory proteins in two different Eubacteria, which both show clear resemblances to the rhodopsins, will be presented. The photoactive yellow protein (PYP) from the purple phototrophic organism Ectothiorhodospira halophila probably functions as the photoreceptor for a new type of negative phototaxis response and has been studied in some detail with respect to its structural and photochemical characteristics. On basis of crystallographic an photochemical data it has been proposed that PYP contains retinal as a chromophore. However, we have unambiguously demonstrated that the PYP chromophore is different from retinal, in spite of the fact that PYP's photochemical properties show striking similarities with the rhodopsins. The cyanobacterium Calothrix sp. displays complementary chromatic adaptation, a process in which the pigment composition of the phycobilisomes is adjusted to the spectral characteristics of the incident light. In orange light the blueish chromophore phycocyanin is present, in green light the reddish phycoerythrin is synthesized. On the basis of the action spectrum of this adaptation process, we hypothesized that a rhodopsin is the photosensor in this process. In line with this, we found that nicotine, an inhibitor of the biosynthesis of beta-carotene (which is the precursor of retinal), abolishes chromatic adaptation. Direct proof of the involvement of a photosensory rhodopsin was obtained in experiments in which the chromatic adaptation response was restored by the addition of retinal to the cultures. The two photosensory proteins mentioned above represent the first examples of eubacterial photoreceptors that can be studied at a molecular level. Our current knowledge on these two proteins and their status as retinal proteins will be reviewed.     

Resonance Raman spectroscopy and quantum chemical calculations reveal structural changes in the active site of photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP(L) intermediate and a long-lived blue-shifted PYP(M) intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP(L) has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP(M), the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP(L) with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP(M) involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.     

Binding, tuning and mechanical function of the 4-hydroxy-cinnamic acid chromophore in photoactive yellow protein.

The bacterial photoreceptor protein photoactive yellow protein (PYP) covalently binds the chromophore 4-hydroxy coumaric acid, tuning (spectral) characteristics of this cofactor. Here, we study this binding and tuning using a combination of pointmutations and chromophore analogs. In all photosensor proteins studied to date the covalent linkage of the chromophore to the apoprotein is dispensable for light-induced catalytic activation. We analyzed the functional importance of the covalent linkage using an isosteric chromophore-protein variant in which the cysteine is replaced by a glycine residue and the chromophore by thiomethyl-p-coumaric acid (TMpCA). The model compound TMpCA is shown to weakly complex with the C69G protein. This non-covalent binding results in considerable tuning of both the pKa and the color of the chromophore. The photoactivity of this system, however, was strongly impaired, making PYP the first known photosensor protein in which the covalent linkage of the chromophore is of paramount importance for the functional activity of the protein in vitro. We also studied the influence of chromophore analogs on the color and photocycle of PYP, not only in WT, but especially in the E46Q mutant, to test if effects from both chromophore and protein modifications are additive. When the E46Q protein binds the sinapinic acid chromophore, the color of the protein is effectively changed from yellow to orange. The altered charge distribution in this protein also results in a changed pKa value for chromophore protonation, and a strongly impaired photocycle. Both findings extend our knowledge of the photochemistry of PYP for signal generation.     

Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein

The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.     

The photoactivated PYP domain of Rhodospirillum centenum Ppr accelerates the recovery of the bacteriophytochrome domain after white light illumination

Ppr from the purple phototrophic bacterium, Rhodospirillum centenum (also known as Rhodocista centenaria), is a hybrid of photoactive yellow protein (PYP), bacteriophytochrome (Bph), and histidine kinase (HK) domains. The holo-Ppr (containing both chromophores) exhibits characteristic absorption maxima at 435 nm due to the PYP domain and at 400, 642, and 701 nm due to the Bph domain. Illumination of the Ppr with white light causes a bleach of both PYP and Bph absorbance; weak blue light primarily bleaches the PYP, and red light activates only the Bph. When excited by blue light, the PYP domain in Ppr recovers with biphasic kinetics at 445 nm (32% with a lifetime of 3.8 min and the remainder with a lifetime of 46 min); white light primarily results in fast recovery, whereas the 130-residue PYP construct shows only the faster kinetics in both blue and white light. Furthermore, there is a slight red shift of the ground state Bph when the PYP is activated; thus, both spectroscopy and kinetics suggest interdomain communication. When Ppr is illuminated with red light, the recovery of the Bph domain to the dark state is significantly slower than that of PYP and is biphasic (57% of the 701 nm decay has a lifetime of 17 min and the remainder a lifetime of 50 min). However, when illuminated with white light or red followed by blue light, the Bph domain in Ppr recovers to the dark-adapted state in a triphasic fashion, where the fastest phase is similar to that of the fast phase of the PYP domain (in white light, 25% of the 701 nm recovery has a lifetime of approximately 1 min) and the slower phases are like the recovery after red light alone. Apo-holo-Ppr (with the biliverdin chromophore only) recovers with biphasic kinetics similar to those of the slower phases of holo-Ppr when activated by either red or white light. We conclude that the photoactivated PYP domain in Ppr accelerates recovery of the activated Bph domain. Phytochromes can be reversibly switched between Pr and Pfr forms by red and far-red light, but the consequence of a bleaching phytochrome is that it cannot be photoreversed by far-red light. We thus postulate that the function of the PYP domain in Ppr is to act as a blue light switch to reverse the effects of red light on the Bph.     

Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in 相似文献   

Light induces destabilization of photoactive yellow protein

To understand the effect of visible light on the stability of photoactive yellow protein (PYP), urea denaturation experiments were performed with PYP in the dark and with PYP(M) under continuous illumination. The urea concentrations at the midpoint of denaturation were 5.26 +/- 0.29 and 3.77 +/- 0.19 M for PYP and PYP(M), respectively, in 100 mM acetate buffer, and 5.26 +/- 0.24 and 4.11 +/- 0.12 M for PYP and PYP(M), respectively, in 100 mM citrate buffer. The free energy change upon denaturation (DeltaG(D)(H2O)), obtained from the denaturation curve, was 11.0 +/- 0.4 and 7.6 +/- 0.2 kcal/mol for PYP and PYP(M), respectively, in acetate buffer, and 11.5 +/- 0.3 and 7.8 +/- 0.1 kcal/mol for PYP and PYP(M), respectively, in citrate buffer. Even though the DeltaG(D)(H2O) value for PYP(M) is almost identical in the two buffer systems, the urea concentration at the midpoint of denaturation is lower in acetate buffer than in citrate buffer. Although their CD spectra indicate that the protein conformations of the denatured states of PYP and PYP(M) are indistinguishable, the configurations of the chromophores in their denatured structures are not necessarily identical. Both denatured states are interconvertible through PYP and PYP(M). Therefore, the free energy difference between PYP and PYP(M) is 3.4-3.7 kcal/mol for the protein moiety, plus the additional contribution from the difference in configuration of the chromophore.     

Spectral Tuning of the Photoactive Yellow Protein Chromophore by H-Bonding

Spectral tuning in the photoactive yellow protein (PYP) is investigated by performing gas-phase absorption measurements on a PYP-model chromophore with two water molecules hydrogen-bonded to it. The photoabsorption maximum shows an unusually large blue shift of 0.71 eV in going from the bare to the hydrogen-bonded chromophore. It is concluded that several interactions within the PYP protein are mutually canceling each other, yielding an absorption maximum that is close to the absorption maximum of the bare chromophore. The system breaks apart upon photoexcitation in the gas phase by releasing the two water molecules, leaving the chromophore itself intact. The hydrogen-bonding interactions thus play an important role in stabilizing the gas phase chromophore against photofragmentation. The relaxation dynamics for the breakup process was also studied, and the timescale of relaxation via fragmentation was found to be <25 ns.     

Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli

Many cyanobacteria use brilliantly pigmented, multisubunit macromolecular structures known as phycobilisomes as antenna to enhance light harvesting for photosynthesis. Recent studies have defined the enzymes that synthesize phycobilin chromophores as well as many of the phycobilin lyase enzymes that attach these chromophores to their cognate apoproteins. The ability of the phycocyanin α-subunit (CpcA) to bind alternative linear tetrapyrrole chromophores was examined through the use of a heterologous expression system in Escherichia coli. E. coli strains produced phycocyanobilin, phytochromobilin, or phycoerythrobilin when they expressed 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), 3Z-phytochromobilin:ferredoxin oxidoreductase (HY2) from Arabidopsis thaliana, or phycoerythrobilin synthase (PebS) from the myovirus P-SSM4, respectively. CpcA from Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 was coexpressed in these strains with the phycocyanin α-subunit phycocyanobilin lyase, CpcE/CpcF, or the phycoerythrocyanin α-subunit phycocyanobilin isomerizing lyase, PecE/PecF, from Noctoc sp. PCC 7120. Both lyases were capable of attaching three different linear tetrapyrrole chromophores to CpcA; thus, up to six different CpcA variants, each with a unique chromophore, could be produced with this system. One of these chromophores, denoted phytoviolobilin, has not yet been observed naturally. The recombinant proteins had unexpected and potentially useful properties, which included very high fluorescence quantum yields and photochemical activity. Chimeric lyases PecE/CpcF and CpcE/PecF were used to show that the isomerizing activity that converts phycocyanobilin to phycoviolobilin resides with PecF and not PecE. Finally, spectroscopic properties of recombinant phycocyanin R-PCIII, in which the CpcA subunits carry a phycoerythrobilin chromophore, are described.     

Resonance Raman evidence for two conformations involved in the L intermediate of photoactive yellow protein

The blue light receptor photoactive yellow protein (PYP) displays a photocycle that involves several intermediate states. Here we report resonance Raman spectroscopic investigations of the short-lived red-shifted intermediate denoted PYP(L). We have found that the Raman bands of the carbonyl C=O stretching mode nu(11) as well as the C=C stretching mode nu(13) for the chromophore can be resolved into two peaks, and the ratio of the two components varies as a function of pH with pK(a) approximately 6. The isotope effects on the resonance Raman spectra have confirmed a deprotonated cis-chromophore for the two components. The results indicate the presence of two conformations in the active site of PYP(L). The normal coordinate calculations based on the density functional theory provide a structural model for the two conformations, where the low pH form is possibly an active structure for the protonation reaction generating a following intermediate in the photocycle.     

Early molecular events in the photoactive yellow protein: role of the chromophore photophysics.

We report a comparative study of the isomerization reaction in native and denatured photoactive yellow protein (PYP) and in various chromophore analogues in their trans deprotonated form. The excited-state relaxation dynamics was followed by subpicosecond transient absorption and gain spectroscopy. The free p-hydroxycinnamate (pCA(2-)) and its amide analogue (pCM(-)) are found to display a quite different transient spectroscopy from that of PYP. The excited-state deactivation leads to the formation of the ground-state cis isomer without any detectable intermediate with a mechanism comparable to trans-stilbene photoisomerization. On the contrary, the early stage of the excited-state deactivation of the free thiophenyl-p-hydroxycinnamate (pCT(-)) and of the denatured PYP is similar to that of the native protein. It involves the formation of an intermediate absorbing in the spectral region located between the bleaching and gain bands in less than 2 ps. However, in these two cases, the formation of the cis isomer has not been proved yet. This difference with pCA(-) and pCM(-) might result from the fact that, in the thioester substituted chromophore, simultaneous population of two quasi-degenerate excited states occurs upon excitation. This comparative study highlights the determining role of the chromophore structure and of its intrinsic properties in the primary molecular events in native PYP.     

Photoactive yellow protein, bacteriophytochrome, and sensory rhodopsin in purple phototrophic bacteria.

The purple photosynthetic bacteria contain a large variety of sensory and regulatory proteins, and those responding to light are among the most interesting. These currently include bacteriophytochrome (Bph), sensory rhodopsin (SR), and photoactive yellow protein (PYP), which all appear to function as light sensors. We herein interpret new findings within the context of current knowledge. For greater detail, the reader is referred to comprehensive reviews on these topics. Of the three proteins, only PYP has been well-characterized in terms of structure and physical-chemical properties in the purple bacteria, although none have well-defined functions. New findings include a cluster of six genes in the Thermochromatium tepidum genome that encodes presumed sensory rhodopsin and phototaxis proteins. T. tepidum also has a gene for PYP fused to bacteriophytochrome and diguanylate cyclase domains. The genes for PYP and its biosynthetic enzymes are associated with those for gas vesicle formation in Rhodobacter species, suggesting that one function of PYP is to regulate cell buoyancy. The association of bacteriophytochrome genes with those for reaction centers and light-harvesting proteins in Rhodopseudomonas palustris suggests that the photosynthetic antenna as well as the reaction center are regulated by Bphs. Furthermore, Rc. centenum PPR is reversibly photobleached at 702 nm rather than red-shifted as in other phytochromes, suggesting that PPR senses the intensity of white light rather than light quality. PYP from Halorhodospira(aka Ectothiorhodospira)halophila is of special interest because it has become the structural prototype for the PAS domain, a motif that is found throughout the phylogenetic tree and which plays important roles in many signaling pathways. Thus, the structural and photochemical characterization of PYP, utilizing site-directed mutagenesis, provides insights into the mechanism of signal transduction.     

Modulating LOV domain photodynamics with a residue alteration outside the chromophore binding site

Phototropins, a class of light-activated protein kinases, are essential for several blue light responses in plants and algae, including phototropism. These proteins contain two internal light, oxygen, and voltage sensitive (LOV) domains, which bind flavin chromophores and undergo a reversible photochemical formation of a cysteinyl-flavin adduct as part of the light sensing process. While the photodynamic properties of such photosensory domains are dictated by interactions between the chromophore and surrounding protein, more distant residues can play a significant role as well. Here we explore the role of the Phe434 residue in the photosensory response of the second LOV domain of Avena sativa phototropin 1 (AsLOV2), a model photochemical system for these LOV domains. Phe434 is more than 6 ? from the FMN chromophore in AsLOV2; nevertheless, an F434Y point mutation is likely to change several structural features of the chromophore binding site, as we demonstrate using molecular dynamics simulations. Transient absorption signals spanning 15 decades in time were compared for wild-type AsLOV2 and the F434Y mutant, showing that the latter has significantly altered photodynamics, including (i) a faster intersystem crossing leading to triplet formation on a nanosecond time scale, (ii) biphasic formation of adduct-state kinetics on the microsecond time scale, and (iii) greatly accelerated ground-state recovery kinetics on a second time scale. We present mechanistic models that link these spectroscopic differences to changes in the configuration of the critical cysteine residue and in the chromophore's accessibility to solvent and oxygen according to MD trajectories and purging experiments. Taken together, these results demonstrate the importance of residues outside the chromophore-binding pocket in modulating LOV domain photodynamics.     

The crystal structure of the R52Q mutant demonstrates a role for R52 in chromophore pKa regulation in photoactive yellow protein

Mutating arginine 52 to glutamine (R52Q) in photoactive yellow protein (PYP) increases the pK(a) of the chromophore by 1 pH unit. The structure of the R52Q PYP mutant was determined by X-ray crystallography and was compared to the structure of wild-type PYP to assess the role of R52 in pK(a) regulation. The essential differences between R52Q and the wild type were confined to the loop region containing the 52nd residue. While the hydrogen bonds involving the chromophore were unchanged by the mutation, removing the guanidino group generated a cavity near the chromophore; this cavity is occupied by two water molecules. In the wild type, R52 forms hydrogen bonds with T50 and Y98; these hydrogen bonds are lost in R52Q. Q52 is linked to Y98 by hydrogen bonding through the two water molecules. R52 acts as a lid on the chromophore binding pocket and controls the accessibility of the exterior solvent and the pK(a) of the chromophore. R52 is found to flip out during the formation of PYP(M). The result of this movement is quite similar to the altered structure of R52Q. Thus, we propose that conformational changes at R52 are partly responsible for pK(a) regulation during the photocycle.     

Resonance Raman spectroscopy reveals the origin of an intermediate wavelength form in photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial blue light receptor containing a 4-hydroxycinnamyl chromophore, and its absorption maximum is 446 nm. In a dark state, the hydroxyl group of the chromophore is deprotonated and forms hydrogen bonds with Tyr42 and Glu46. Either removal of a hydrogen bond with Tyr42 or addition of chaotropes such as thiocyanate produces a blue-shifted species called an intermediate wavelength form, in which absorption maximum ranges from 355 to 400 nm. To examine the structural origin of the intermediate wavelength form, we have performed resonance Raman investigations of wild-type PYP and some mutants (Tyr42 --> Ala, Tyr42 --> Phe, Glu46 --> Gln, and Thr50 --> Val) in the presence or absence of potassium thiocyanate. These studies show that the chromophore of the intermediate wavelength form is protonated, implying an increase in a pK(a) of the chromophore. Hence, the removal of the hydrogen bond between Tyr42 and chromophore or partial protein denaturation in the presence of thiocyanate results in a spectral blue-shift. Quantum chemical calculations based on density functional theory further support the idea that the pK(a) of the chromophore is increased by removing a hydrogen bond or by increasing the dielectric constant in the vicinity of the chromophore.     

Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein

To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.     

Predicting the signaling state of photoactive yellow protein

As a bacterial blue light sensor the photoactive yellow protein (PYP) undergoes conformational changes upon signal transduction. The absorption of a photon triggers a series of events that are initially localized around the protein chromophore, extends to encompass the whole protein within microseconds, and leads to the formation of the transient pB signaling state. We study the formation of this signaling state pB by molecular simulation and predict its solution structure. Conventional straightforward molecular dynamics is not able to address this formation process due to the long (microsecond) timescales involved, which are (partially) caused by the presence of free energy barriers between the metastable states. To overcome these barriers, we employed the parallel tempering (or replica exchange) method, thus enabling us to predict qualitatively the formation of the PYP signaling state pB. In contrast to the receptor state pG of PYP, the characteristics of this predicted pB structure include a wide open chromophore-binding pocket, with the chromophore and Glu(46) fully solvent-exposed. In addition, loss of alpha-helical structure occurs, caused by the opening motion of the chromophore-binding pocket and the disruptive interaction of the negatively charged Glu(46) with the backbone atoms in the hydrophobic core of the N-terminal cap. Recent NMR experiments agree very well with these predictions.     

Application of photoactivatable fluorescent active-site directed probes to serine-containing enzymes

A photoactivatable fluorescent anthraniloyl group has been directed to the active-site serine group of alpha-chymotrypsin and trypsin. The acylated derivatives are nonfluorescent until irradiated. When activated by light a highly reactive nitrene is generated which is capable of covalent insertion into the protein matrix. The resultant insertion product of this photolysis is a highly fluorescent reporter group which has little rotational mobility and is cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked enzyme and deacylated enzyme were determined. These measurements show that little relaxation occurs on the nanosecond time scale for the cross-linked enzyme, but upon deacylation of the serine increased dipolar relaxation of the protein with the attached reporter group is observed. The use of these active-site directed photoactivatable fluorescent probes can be extended to probe the active-site structure of complex enzymes and conformational dynamics of active-site regions in proteins and to serve as potential functional site labels in fluorescence resonance energy transfer measurements.     

Mimic of photocycle by a protein folding reaction in photoactive yellow protein

The blue light receptor photoactive yellow protein (PYP) displays rhodopsin-like photochemistry based on the trans to cis photoisomerization of its p-coumaric acid chromophore. Here, we report that protein refolding from the acid-denatured state of PYP mimics the last photocycle transition in PYP. This implies a direct link between transient protein unfolding and photosensory signal transduction. We utilize this link to study general issues in protein folding. Chromophore trans to cis photoisomerization in the acid-denatured state strongly decelerates refolding, and converts the pH dependence of the barrier for refolding from linear to nonlinear. We propose transition state movement to explain this phenomenon. The cis chromophore significantly stabilizes the acid-denatured state, but acidification of PYP results in the accumulation of the acid-denatured state containing a trans chromophore. This provides a clear example of kinetic control in a protein unfolding reaction. These results demonstrate the power of PYP as a light-triggered model system to study protein folding.     

Function and structure of GFP-like proteins in the protein data bank

The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the β-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in β-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.