Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue-shifted intermediate

The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.     

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.     

Light-induced unfolding of photoactive yellow protein mutant M100L

Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563-11568]. The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M). The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M). The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M). These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.     

Structural characterization of the M* partly folded intermediate of wild type and P138A aspartate aminotransferase from Escherichia coli

A combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M* partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M* intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N(2) forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposable.     

Rational design, structural and thermodynamic characterization of a hyperstable variant of the villin headpiece helical subdomain

A hyperstable variant of the small independently folded helical subdomain (HP36) derived from the F-actin binding villin headpiece was designed by targeting surface electrostatic interactions and helical propensity. A double mutant N68A, K70M was significantly more stable than wild type. The Tm of wild type in aqueous buffer is 73.0 degrees C, whereas the double mutant did not display a complete unfolding transition. The double mutant could not be completely unfolded even by 10 M urea. In 3 M urea, the Tm of wild type is 54.8 degrees C while that of the N68AK70M double mutant is 73.9 degrees C. Amide H/2H exchange studies show that the pattern of exchange is very similar for wild type and the double mutant. The structures of a K70M single mutant and the double mutant were determined by X-ray crystallography and are identical to that of the wild type. Analytical ultracentrifugation demonstrates that the proteins are monomeric. The hyperstable mutant described here is expected to be useful for folding studies of HP36 because studies of the wild type domain have sometimes been limited by its marginal stability. The results provide direct evidence that naturally occurring miniature protein domains have not been evolutionarily optimized for global stability. The stabilizing effect of this double mutant could not be predicted by sequence analysis because K70 is conserved in the larger intact headpiece for functional reasons.     

Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr

Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pK_a is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pK_a shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.     

Helix formation is a dynamical bottleneck in the recovery reaction of Photoactive Yellow Protein

The bacterial sensor Photoactive Yellow Protein (PYP) signals the presence of blue light by undergoing a series of conformational changes. We present atomistic Parallel Tempering (Replica Exchange Molecular Dynamics) simulations of conformational changes occurring during the photo-cycle of PYP. First, we study the signaling state formation of PYP in detail. Our previous simulations have shown that the formation of the signaling state is characterized by the solvent exposure of both the chromophore and Glu46 (Vreede J, Crielaard W, Hellingwerf KJ, Bolhuis PG. Biophys J, 2005;8:3525-3535). Subsequent NMR results agreed with this prediction, but as these experiments were performed on an N-terminally truncated mutant, a simulation of this mutant would further substantiate our previous results. Here, we compare simulations of the truncated PYP to the NMR structures, as well as to the wild type predictions. This comparison also gives some insight into the role of the N-terminal domain of PYP, which restricts the movement of the chromophore binding pocket (CBP) in the wild type. Second, we report simulations of the recovery of the receptor state from the signaling state. While we did not observe complete refolding of the protein, we did observe transient interactions between residues of the CBP occurring when the chromophore is in a trans configuration. Using simulations that sample anomalous exposure of the chromophore in the receptor state, we were able to sample chromophore re-entry into its binding pocket. While the involved time scales prohibit drawing definitive conclusions even when using parallel tempering, we nevertheless propose that the formation of a helix in the CBP is essential for a successful recovery of the receptor state, and forms a kinetic barrier in this process.     

Intrinsic local destabilization of the C‐terminus predisposes integrin α1 I domain to a conformational switch induced by collagen binding

Integrin–collagen interactions play a critical role in a myriad of cellular functions that include immune response, and cell development and differentiation, yet their mechanism of binding is poorly understood. There is increasing evidence that conformational flexibility assumes a central role in the molecular mechanisms of protein–protein interactions and here we employ NMR hydrogen–deuterium exchange (HDX) experiments to explore the impact of slower timescale dynamic events. To gain insight into the mechanisms underlying collagen‐induced conformational switches, we have undertaken a comparative study between the wild type integrin α1 I and a gain‐of‐function E317A mutant. NMR HDX results suggest a relationship between regions exhibiting a reduced local stability in the unbound I domain and those that undergo significant conformational changes upon binding. Specifically, the αC and α7 helices within the C‐terminus are at the center of such major perturbations and present reduced local stabilities in the unbound state relative to other structural elements. Complementary isothermal titration calorimetry experiments have been performed to derive complete thermodynamic binding profiles for association of the collagen‐like triple‐helical peptide with wild type α1 I and E317A mutant. The differential energetics observed for E317A are consistent with the HDX experiments and support a model in which intrinsically destabilized regions predispose conformational rearrangement in the integrin I domain. This study highlights the importance of exploring different timescales to delineate allosteric and binding events.     

Consequences of counterion mutation in sensory rhodopsin II of Natronobacterium pharaonis for photoreaction and receptor activation: an FTIR study

In many retinal proteins the proton transfer from the Schiff base to the counterion represents a functionally important step of the photoreaction. In the signaling state of sensory rhodopsin II from Natronobacterium pharaonis this transfer has already occurred, but in the counterion mutant Asp75Asn it is blocked during all steps of the photocycle. Therefore, the study of the molecular changes during the photoreaction of this mutant should provide a deeper understanding of the activation mechanism, and for this, we have applied time-resolved step-scan FTIR spectroscopy. The photoreaction is drastically altered; only red-shifted intermediates are formed with a chromophore strongly twisted around the 14-15 single bond. In addition, the photocycle is shortened by 2 orders of magnitude. Nevertheless, a transition involving only protein changes similar to that of the wild type is observed, which has been correlated with the formation of the signaling state. However, whereas in the wild type this transition occurs in the millisecond range, it is shortened to 200 micros in the mutant. The results are discussed with respect to the altered electrostatic interactions, role of proton transfer, the published 3D structure, and physiological activity.     

The angles between the C(1)-, C(5)-, and C(9)-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: direct determination with solid-state (2)H NMR.

The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR ((2)H NMR). In this key intermediate, the chromophore has a 13-cis, 15-anti conformation and a deprotonated Schiff base. Purple membranes containing wild-type or mutant D96A bacteriorhodopsin were regenerated with retinals specifically deuterated in the methyl groups of either carbon C(1) or C(5) of the beta-ionone ring or carbon C(9) of the polyene chain. Oriented hydrated films were formed by drying concentrated suspensions on glass plates at 86% relative humidity. The lifetime of the M state was increased in the wild-type samples by applying a guanidine hydrochloride solution at pH 9.5 and in the D96A sample by raising the pH. (2)H NMR experiments were performed on the dark-adapted ground state (a 2:1 mixture of 13-cis, 15-syn and all-trans, 15-anti chromophores), the cryotrapped light-adapted state (all-trans, 15-anti), and the cryotrapped M intermediate (13-cis, 15-anti) at -50 degrees C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5 degrees C and then rapidly cooled to -50 degrees C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the (2)H NMR line shapes, the angles between the individual C-CD(3) bonds and the membrane normal could be determined even in the presence of a substantial degree of orientational disorder. While only minor differences were detected between dark- and light-adapted states, all three angles increase in the M state. This is consistent with an upward movement of the C(5)-C(13) part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C(9)-CD(3) bond shows the largest orientational change of 7 degrees in M. This reorientation of the chromophore in the binding pocket provides direct structural support for previous suggestions (based on spectroscopic evidence) for a steric interaction in M between the C(9)-methyl group and Trp 182 in helix F.     

A role of methionine 100 in facilitating PYP(M)-decay process in the photocycle of photoactive yellow protein

PYP (photoactive yellow protein) is a photoreceptor protein, which is activated upon photo-isomerization of the p-coumaric acid chromophore and is inactivated as the chromophore is thermally back-isomerized within a second (in PYP(M)-to-PYP(dark) conversion). Here we have examined the mechanism of the rapid thermal isomerization by analyzing mutant PYPs of Met100, which was previously shown to play a major role in facilitating the reaction [Devanathan, S. et al. (1998) Biochemistry 37, 11563-11568]. The mutation to Lys, Leu, Ala, or Glu decelerated the dark state recovery by one to three orders of magnitude. By evaluating temperature-dependence of the kinetics, it was found that the retardation resulted unequivocally from elevations of activation enthalpy (DeltaH( double dagger )) but not the other parameters such as activation entropy or heat capacity changes. Another effect exerted by the mutations was an up-shift of the apparent pK(a) of the chromophore [the pK(a) of a titratable group (X) that controls the pK(a) of the chromophore] in the PYP(M)-decay process. The pK(a) up-shift and the DeltaH( double dagger ) elevation show an approximately linear correlation. We, therefore, postulate that the role of Met100 is to reduce the energy barrier of the PYP(M)-decay process by an indirect interaction through X and that the process is thereby facilitated.     

Molecular basis of photochromism of a fluorescent protein revealed by direct <Superscript>13</Superscript>C detection under laser illumination

Dronpa is a green fluorescent protein homologue with a photochromic property. A green laser illumination reversibly converts Dronpa from a green-emissive bright state to a non-emissive dark state, and ultraviolet illumination converts it to the bright state. We have employed solution NMR to understand the underlying molecular mechanism of the photochromism. The detail characterization of Dronpa is hindered as it is metastable in the dark state and spontaneously converts to the bright state. To circumvent this issue, we have designed in magnet laser illumination device. By combining the device with a 150-mW argon laser at 514.5 nm, we have successfully converted and maintained Dronpa in the dark state in the NMR tube by continuous illumination during the NMR experiments. We have employed direct-detection of ¹³C nuclei from the carbon skeleton of the chromophore for detailed characterization of chromophore in both states of Dronpa by using the Bruker TCI cryoprobe. The results from NMR data have provided direct evidence of the double bond formation between C^α and C^β of Y63 in the chromophore, the β-barrel structure in solution, and the ionized and protonated state of Y63 hydroxyl group in the bright and dark states, respectively. These studies have also revealed that a part of β-barrel around the chromophore becomes polymorphic only in the dark state, which may be critical to make the fluorescence dim by increasing the contribution of non-emissive vibrational relaxation pathways.     

Structural role of tyrosine 98 in photoactive yellow protein: effects on fluorescence, gateway, and photocycle recovery

We have recently shown that the Y98Q mutant of PYP has a major effect on the photocycle kinetics ( approximately 40 times slower recovery). We have now determined the crystal structure of Y98Q at 2.2 A resolution to reveal the role of residue Y98 in the PYP photocycle. Although the overall structure is very similar to that of WT, we observed two major effects of the mutation. One obvious consequence is a conformational change of the beta4-beta5 loop, which includes a repositioning of residue M100. It had previously been shown that the photocycle is slowed by as much as 3 orders of magnitude when residue M100 is substituted or when the conformation is altered as in Rhodocista centenaria PYP. To investigate whether the altered photocycle of Y98Q is due to this repositioning of M100 or is caused by an effect unrelated to M100, we determined the dark recovery kinetics of the Y98Q/M100A mutant. We find the recovery kinetics to be very similar to the M100A single mutant kinetics and therefore conclude that the slower recovery kinetics in Y98Q are most likely due to repositioning of M100. In addition, we find that other substitutions at position 98 (Y98W, Y98L, and Y98A) have differing effects on the photocycle recovery, presumably due to a variable distortion of the beta4-beta5 loop. The second effect of the Y98Q mutation is a repositioning of R52, which is thought to interact with Y98 in WT PYP and now forms new interactions with residues Q99 and Q56. To determine the role of R52, we also characterized an R52A/M100A double mutant and found that the effects on the recovery kinetics ( approximately 2000 slower recovery than WT) are due to unrelated events in the photocycle. Since the Y98Q/M100A recovery kinetics are more similar to those of M100 than R52A/M100A, we conclude that the repositioning of R52, caused by the Y98Q mutation, does not affect the dark state recovery. In addition, it has been proposed that Y98 and P68 are "gateway residues" between which the chromophore must pass during isomerization. We tested the recovery kinetics of mutant P68A and found that, although the gateway may be important for photocycle initiation, its role in recovery to the ground state is minimal.     

Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photo-induced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc --> Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.     

Regulation of cyclic electron transport through photosystem I in cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 mutants deficient in respiratory dehydrogenases

The rate of PSI mediated cyclic electron transport was studied in wild type and mutant cells of Synechocystis sp. PCC 6803 deficient in NDH-1 (M55) or succinate dehydrogenase (SDH⁻) that are responsible for the dark reduction of the plastoquinone pool. Kinetics of P700 photooxidation and P700⁺ dark reduction in the presence of 5·10⁻⁵ M 3-(3,4-dichlorophenyl)-1,1-dimethylurea have been registered as light induced absorbance changes at 810 nm resulting from illumination of cells with 730-nm actinic light for 1 sec. It is shown that in the absence of dehydrogenases the rate of dark reduction of P700⁺ in both mutants did not decrease but even increased in NDH-1-less mutant cells as compared with the rate in wild type cells. Dibromothymoquinone drastically reduced the rate of P700⁺ dark reduction both in wild type and in mutant cells. Thus, the cyclic electron transfer from ferredoxin through the plastoquinone pool to P700⁺, which is independent from dehydrogenases, takes place in all the types of cells. Preillumination of cells of wild type and both mutants for 30 min or anaerobic conditions resulted in delay of P700 photooxidation and acceleration of P700⁺ dark reduction, while the level of photosynthesis and respiration terminal acceptors (NAD(P)⁺ and oxygen) decreased. It appears that the rate of P700 photooxidation and P700⁺ dark reduction in cyclic electron transport in Synechocystis wild type and mutant cells is determined by the level of NADP⁺ and oxygen in stroma. A possible approach to evaluation of the levels of these acceptors in vivo is proposed, based on kinetic curve parameters of P700 photoconversions induced by 730-nm light with 1-sec duration.     

Towards an interpretation of 13C chemical shifts in bathorhodopsin,a functional intermediate of a G-protein coupled receptor

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired ¹³C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the ¹³C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the ¹³C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.     

An Investigation of Electron Spin Resonance in Wild Type Chlamydomonas reinhardi and Mutant Strains Having Impaired Photosynthesis

The electron spin resonance signals of wild type Chlamydomonas reinhardi and three mutant strains having impaired photosynthesis have been investigated. The wild type strain generates two different electron spin resonance signals. Signal I is obtained without illumination (i.e., dark signal) whereas signal II is generated preferentially only by red light. Signal I is missing from wild type cells that have been cultured in the dark, but it returns after these dark-grown cells have been illuminated. Chloroplast fragments obtained from the three mutant strains cannot photoreduce TPN. Two of the strains lack the dark signal I while the third strain has both signal I and signal II. Other studies have revealed that the two mutant strains which lack signal I give no Hill reaction but that they can photoreduce TPN if supplied with an artificial reductant. The mutant strain which has both electron spin resonance signals can carry out the Hill reaction, yet it too will not photoreduce TPN unless reductant is supplied. The electron spin resonance signals generated by the wild type and mutant strains are discussed in terms of the pathway of TPN photoreduction, and it is suggested that signal I is associated with one of the two light-dependent phases of this pathway.     

Kindling fluorescent protein from Anemonia sulcata: dark-state structure at 1.38 A resolution

When the nonfluorescent chromoprotein asFP595 from Anemonia sulcata is subjected to sufficiently intense illumination near the absorbance maximum (lambda(abs)(max) = 568 nm), it undergoes a remarkable transition, termed "kindling", to a long-lived fluorescent state (lambda(em)(max) = 595 nm). In the dark recovery phase, the kindled state relaxes thermally on a time scale of seconds or can instantly be reverted upon illumination at 450 nm. The kindling phenomenon is enhanced by the Ala143 --> Gly point mutation, which slows the dark recovery time constant to 100 s at room temperature and increases the fluorescence quantum yield. To investigate the chemical nature of the chromophore and the possible role of chromophore isomerization in the kindling phenomenon, we determined the crystal structure of the "kindling fluorescent protein" asFP595-A143G (KFP) in the dark-adapted state at 1.38 A resolution and 100 K. The chromophore, derived from the Met63-Tyr64-Gly65 tripeptide, closely resembles that of the nonfluorescent chromoprotein Rtms5 in that the configuration is trans about the methylene bridge and there is substantial distortion from planarity. Unlike in Rtms5, in the native protein the polypeptide backbone is cleaved between Cys62 and Met63. The size and shape of the chromophore pocket suggest that the cis isomer of the chromophore could also be accommodated. Within the pocket, partially disordered His197 displays two conformations, which may constitute a binary switch that stabilizes different chromophore configurations. The energy barrier for thermal relaxation was found by Arrhenius plot analysis to be approximately 71 kJ/mol, somewhat higher than the value of approximately 55 kJ/mol observed for cis-trans isomerization of a model chromophore in solution.     

Formation of a long-lived photoproduct with a deprotonated Schiff base in proteorhodopsin, and its enhancement by mutation of Asp227

Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-lived 362 nm photoproduct is apparently a side reaction of the photocycle, a response to high pH, caused by alteration of the normal reprotonation and reisomerization pathway of the Schiff base.     

Mutational Analysis of Dark Endogenous Metabolism in the Blue-Green Bacterium Anacystis nidulans

We describe a mutant (strain 704) of the obligate photoautotroph Anacystis nidulans which behaves like the wild type under continuous illumination but which in the dark rapidly loses viability, respires little, and incorporates label into ribonucleic acid and protein at rates considerably less than observed with the darkened wild type. Extracts of this mutant strain show no detectable 6-phosphogluconate dehydrogenase (EC 1.1.1.44) activity. Spontaneous revertants of mutant 704 were selected as survivors of prolonged incubation in darkness. Of 10 such strains examined, none had regained 6-phosphogluconate dehydrogenase activity, and all had lost detectable glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity. Although dark survival of these revertants paralleled that of the wild type, rates of dark endogenous respiration and incorporation of labeled precursors into ribonucleic acid were still very low, comparable to those observed with strain 704. These results are consistent with the following hypotheses concerning dark endogenous metabolism in unicellular blue-green bacteria. (i) Although the oxidative pentose phosphate cycle (hexose monophosphate shunt) may play a major role in endogenous metabolism in A. nidulans, as proposed by others, it is not the only pathway capable of providing energy for maintenance of viability in darkness. (ii) Much of the endogenous metabolic activity (respiration and macromolecular synthesis) observed in darkened cultures of wild-type A. nidulans is not required for survival alone, and must therefore serve other functions.