Secondary literature sources for PAC

The following references were automatically generated.

Shiozawa M et al.
Evidence for large structural fluctuations of the photobleached intermediate of photoactive yellow protein in solution.
J Am Chem Soc. 2001; 123: 7445-6

Molina V, Merchan M
On the absorbance changes in the photocycle of the photoactive yellow protein: a quantum-chemical analysis.
Proc Natl Acad Sci U S A. 2001; 98: 4299-304
Display abstract
Spectral changes in the photocycle of the photoactive yellow protein (PYP) are investigated by using ab initio multiconfigurational second-order perturbation theory at the available structures experimentally determined. Using the dark ground-state crystal structure [Genick, U. K., Soltis, S. M., Kuhn, P., Canestrelli, I. L. & Getzoff, E. D. (1998) Nature (London) 392, 206-209], the pipi* transition to the lowest excited state is related to the typical blue-light absorption observed at 446 nm. The different nature of the second excited state (npi*) is consistent with the alternative route detected at 395-nm excitation. The results suggest the low-temperature photoproduct PYP(HL) as the most plausible candidate for the assignment of the cryogenically trapped early intermediate (Genick et al.). We cannot establish, however, a successful correspondence between the theoretical spectrum for the nanosecond time-resolved x-ray structure [Perman, B., Srajer, V., Ren, Z., Teng, T., Pradervand, C., et al. (1998) Science 279, 1946-1950] and any of the spectroscopic photoproducts known up to date. It is fully confirmed that the colorless light-activated intermediate recorded by millisecond time-resolved crystallography [Genick, U. K., Borgstahl, G. E. O., Ng, K., Ren, Z., Pradervand, C., et al. (1997) Science 275, 1471-1475] is protonated, nicely matching the spectroscopic features of the photoproduct PYP(M). The overall contribution demonstrates that a combined analysis of high-level theoretical results and experimental data can be of great value to perform assignments of detected intermediates in a photocycle.

Devanathan S, Lin S, Cusanovich MA, Woodbury N, Tollin G
Early photocycle kinetic behavior of the e46a and y42f mutants of photoactive yellow protein: femtosecond spectroscopy.
Biophys J. 2001; 81: 2314-9
Display abstract
In the photoactive yellow protein, PYP, both Glu46 and Tyr42 form hydrogen bonds to the phenolic OH group of the p-hydroxycinnamoyl chromophore. Previous work on replacement of the carboxyl group of Glu46 by an amide group (Glu46Gln) has shown that changing the nature of this hydrogen bond has a minimal effect on the rate constant for the formation of the first intermediate (I(0)) and on the excited state lifetime, whereas the rate constants for the formation of the second (I(0)( not equal)) and third (I(1)) intermediates were increased by factors of ~30 and 5, respectively. In the present experiments, two additional mutants (Glu46Ala and Tyr42Phe) have been studied. These two mutants are shown to behave kinetically very similarly to one another. In both cases, the rate constant for I(0) formation is decreased by a factor of ~2, with little or no effect on the photochemical yield as a consequence of a compensating increase in the excited state lifetime. Although we are unable to resolve the rate constant for the formation of the second intermediate from that of the first intermediate, the rate constant for the formation of the third intermediate is increased by a factor of ~100. The structural implications of these results are discussed.

Xie A, Kelemen L, Hendriks J, White BJ, Hellingwerf KJ, Hoff WD
Formation of a new buried charge drives a large-amplitude protein quake in photoreceptor activation.
Biochemistry. 2001; 40: 1510-7
Display abstract
Photoactive yellow protein (PYP) is a eubacterial photoreceptor and a structural prototype of the PAS domain superfamily of receptor and regulatory proteins. We investigate the activation mechanism of PYP using time-resolved Fourier transform infrared (FTIR) spectroscopy. Our data provide structural, kinetic, and energetic evidence that the putative signaling state of PYP is formed during a large-amplitude protein quake that is driven by the formation of a new buried charge, COO(-) of the conserved Glu46, in a highly hydrophobic pocket at the active site. A protein quake is a process consisting of global conformational changes that are triggered and driven by a local structural "fault". We show that large, global structural changes take place after Glu46 ionization via intramolecular proton transfer to the anionic p-coumarate chromophore, and are suppressed by the absence of COO(-) formation in the E46Q mutant. Our results demonstrate the significance of buried charge formation in photoreceptor activation. This mechanism may serve as one of the general themes in activation of a range of receptor proteins. In addition, we report the results of time-resolved FTIR spectroscopy of PYP crystals. The direct comparison of time-resolved FTIR spectroscopic data of PYP in aqueous solution and in crystals reveals that the structure of the putative signaling state is not developed in P6(3) crystals. Therefore, when the structural developments during the functional process of a protein are experimentally determined to be very different in crystals and solutions, one must be cautious in drawing conclusions regarding the functional mechanism of proteins based on time-resolved X-ray crystallography.

Imamoto Y et al.
Roles of amino acid residues near the chromophore of photoactive yellow protein.
Biochemistry. 2001; 40: 4679-85
Display abstract
To investigate the roles of amino acid residues around the chromophore in photoactive yellow protein (PYP), new mutants, Y42A, E46A, and T50A were prepared. Their spectroscopic properties were compared with those of wild-type, Y42F, E46Q, T50V, R52Q, and E46Q/T50V, which were previously prepared and specified. The absorption maxima of Y42A, E46A, and T50A were observed at 438, 469, and 454 nm, respectively. The results of pH titration for the chromophore demonstrated that the chromophore of PYP mutant, like the wild-type, was protonated and bleached under acidic conditions. The red-shifts of the absorption maxima in mutants tended toward a pK(a) increase. Mutation at Glu46 induced remarkable shifts in the absorption maxima and pK(a). The extinction coefficients were increased in proportion to the absorption maxima, whereas the oscillator strengths were constant. PYP mutants that conserved Tyr42 were in the pH-dependent equilibrium between two states (yellow and colorless forms). However, Y42A and Y42F were in the pH-independent equilibrium between additional intermediate state(s) at around neutral pH, in which yellow form was dominant in Y42F whereas the other was dominant in Y42A. These findings suggest that Tyr42 acts as the hinge of the protein, and the bulk as well as the hydroxyl group of Tyr42 controls the protein conformation. In all mutants, absorbance at 450 nm was decreased upon flash irradiation and afterwards recovered on a millisecond time scale. However, absorbance at 340--370 nm was increased vice versa, indicating that the long-lived near-UV intermediates are formed from mutants, as in the case of wild-type. The lifetime changes with mutation suggest the regulation of proton movement through a hydrogen-bonding network.

Harigai M, Yasuda S, Imamoto Y, Yoshihara K, Tokunaga F, Kataoka M
Amino acids in the n-terminal region regulate the photocycle of photoactive yellow protein.
J Biochem (Tokyo). 2001; 130: 51-6
Display abstract
The spectroscopic properties of photoactive yellow protein (PYP) partially digested by chymotrypsin were studied. Chymotrypsin yielded three major products that were yellow but distinguishable by SDS-PAGE. They were readily separated by DEAE-Sepharose column chromatography. Protein sequencing and mass spectrometry demonstrated that chymotrypsin cleaved the N-terminal 6, 15, or 23 amino acids (T6, T15, and T23). The blue-shifts of the absorption maxima and the increases in the apparent pK(a) of the chromophores relative to those of intact PYP were less than 4 nm and 0.2, respectively. The absorption spectra of the near-UV intermediates produced from T6, T15, and T23 were identical to that of intact PYP, but with lifetimes that were 140, 2,300, and 4,500 times longer, respectively. These observations suggest that the recovery of the dark state of PYP from the near-UV intermediate is accelerated by the N-terminal region, and that this region acts as a regulatory factor for the photocycle of PYP.

van der Horst MA, van Stokkum IH, Crielaard W, Hellingwerf KJ
The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation.
FEBS Lett. 2001; 497: 26-30
Display abstract
It is shown that the N-terminal domain of photoactive yellow protein (PYP), which appears relatively independently folded in the ground state of the protein, plays a key role in the transient unfolding during signalling state formation: genetic truncation of the N-terminal domain of PYP significantly decreases the extent of cooperativity of the titration curve that describes chromophore protonation in the ground state of PYP, which is in agreement with the notion that the N-terminal domain is linked through a hydrogen-bonding network with the chromophore-containing domain of the protein. Furthermore, deletion of the N-terminal domain completely abolishes the non-linearity of the Arrhenius plot of the rate of ground state recovery.

Hassan BA, Bellen HJ
Doing the MATH: is the mouse a good model for fly development?
Genes Dev. 2000; 14: 1852-65

Hogenesch JB et al.
The basic helix-loop-helix-PAS protein MOP9 is a brain-specific heterodimeric partner of circadian and hypoxia factors.
J Neurosci. 2000; 20: 83-83
Display abstract
PAS (PER, ARNT, SIM) proteins play important roles in adaptation to low atmospheric and cellular oxygen levels, exposure to certain environmental pollutants, and diurnal oscillations in light and temperature. In an attempt to better understand how organisms sense environmental changes, we have characterized a novel member of the PAS superfamily, MOP9 (member of PAS superfamily), that maps to human chromosome 12p11.22-11.23. This protein displays significant homology to the Drosophila circadian factor CYCLE and its putative mammalian ortholog MOP3/bMAL1. Like its homologs, MOP9 forms a transcriptionally active heterodimer with the circadian CLOCK protein, the structurally related MOP4, and hypoxia-inducible factors, such as HIF1alpha. In a manner consistent with its role as a biologically relevant partner of these proteins, MOP9 is coexpressed in regions of the brain such as the thalamus, hypothalamus, and amygdala. Importantly, MOP9 is coexpressed with CLOCK in the suprachiasmatic nucleus, the site of the master circadian oscillator in mammals.

Ma Y et al.
Functional interactions between Drosophila bHLH/PAS, Sox, and POU transcription factors regulate CNS midline expression of the slit gene.
J Neurosci. 2000; 20: 4596-605
Display abstract
During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent on Single-minded (Sim), a basic-helix-loop-helix (bHLH)-PAS transcription factor. We show here that Fish-hook (Fish), a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression. single-minded, fish-hook, and drifter are all expressed in developing midline cells, and both loss- and gain-of-function assays revealed genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells. Fish-hook directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Fish can form homodimers and also associates with other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes.

Bao J, Talmage DA, Role LW, Gautier J
Regulation of neurogenesis by interactions between HEN1 and neuronal LMO proteins.
Development. 2000; 127: 425-35
Display abstract
Basic-helix-loop-helix transcription factors regulate neurogenesis and neuronal differentiation by as yet unknown mechanisms. We show that an embryonic neuronal-specific basic-helix-loop-helix protein, HEN1 (also known as NSCL1 or NHLH), interacts with 'LIM only' proteins. Examination of the expression patterns of XHEN1 and XLMO-3, the Xenopus homologues of these human genes, reveals extensive overlap during early neurogenesis: at the onset of gastrulation on the dorsal side of the blastopore lip and, subsequently, in the prospective neural plate. Binding of XLMO-3 increases the transcriptional activity of XHEN1 in vivo. Co-expression of these two genes in Xenopus embryos induces a cascade of expression of neuronal-specific basic-helix-loop-helix proteins that leads to neuronal differentiation. We propose that XHEN1, in concert with XLMO-3, is a critical regulator of neurogenesis.

Goulding SE, White NM, Jarman AP
cato encodes a basic helix-loop-helix transcription factor implicated in the correct differentiation of Drosophila sense organs.
Dev Biol. 2000; 221: 120-31
Display abstract
In Drosophila neurogenesis, proneural genes encode bHLH proteins that are required for neural precursor selection. But many vertebrate homologues are expressed later and are postulated to have multiple roles during neurogenesis. We have isolated a new Drosophila gene, cato, which encodes a protein with a bHLH domain that is closely related to that of the proneural protein Atonal. cato expression is restricted to the developing PNS, where it is expressed in between the stages of precursor selection and terminal differentiation (and therefore later than the proneural genes). We present evidence from loss-of-function and misexpression experiments that cato is involved in sensory neurone morphology. Moreover, in prospero mutants, in which axon and dendrite outgrowth is defective, cato is strongly derepressed in the developing CNS.

Steidl C et al.
Characterization of the human and mouse HEY1, HEY2, and HEYL genes: cloning, mapping, and mutation screening of a new bHLH gene family.
Genomics. 2000; 66: 195-203
Display abstract
Many basic helix-loop-helix (bHLH) transcription factors are known as key regulators of embryonic development or differentiation in various species. We have isolated and characterized three new hairy-related bHLH transcription factor genes from mouse and human (hairy and Enhancer-of-split related with YRPW motif; HEY1, HEY2, and HEYL). All three HEY genes have a similar genomic structure with five exons. Together with a highly related Drosophila homologue, they form a new bHLH gene subfamily that is different from both hairy and the known vertebrate Hes and Her genes. While the overall structure with the bHLH domain, Orange domain, and WRPW motif is similar, the last motif is changed to KPYRPWG in Hey1/2 and absent in HeyL. This and other sequence features suggest Hey proteins to have unique functional properties. The genes were mapped by fluorescence in situ hybridization and RH mapping to the following human chromosomes: (HEY1) 8q21, (HEY2) 6q21, and (HEYL) 1p34.3. Based on expression patterns and map location, HEY genes are candidates for several human or mouse disease loci. However, initial screening of DNA from affected individuals for two human disorders and four mouse mutants did not reveal any diagnostic alterations in the coding regions.

Lewis RJ, Muchova K, Brannigan JA, Barak I, Leonard G, Wilkinson AJ
Domain swapping in the sporulation response regulator Spo0A.
J Mol Biol. 2000; 297: 757-70
Display abstract
Adaptive responses of micro-organisms, such as chemotaxis and sporulation, are governed by two-component systems consisting of sensor kinases, that interpret environmental signals, and response regulators which activate the appropriate physiological responses. Signal transduction via response regulator proteins is mediated through transient phosphorylation of aspartic acid residues. In Spo0A, the key regulator of development (sporulation) in Bacillus, phosphorylation of the N-terminal receiver domain (N-Spo0A) at aspartate-55 switches on the transcription activation functions residing in the C-terminal effector domain. Here we report the crystal structure of N-Spo0A from Bacillus stearothermophilus at 1.6 A spacing, revealing a dimer formed by an alpha-helix swap. Comparison of this structure with the recently described structure of phosphorylated N-Spo0A shows that dimer formation results from a cis-trans isomerization of the Lys106--Pro107 peptide bond. The quaternary reorganization is associated with alterations in the active site stereochemistry which may have implications for signalling. Remarkably, this 3-D domain swapped N-Spo0A dimer has an identical topology to a hypothetical CheY-like dimer, recently proposed as an intermediate in the evolution of the family of periplasmic substrate binding proteins.

Taylor BL, Zhulin IB
PAS domains: internal sensors of oxygen, redox potential, and light.
Microbiol Mol Biol Rev. 1999; 63: 479-506
Display abstract
PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells.

Gouet P et al.
Structural transitions in the FixJ receiver domain.
Structure Fold Des. 1999; 7: 1517-26
Display abstract
BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.

Englund C, Uv AE, Cantera R, Mathies LD, Krasnow MA, Samakovlis C
adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS.
Development. 1999; 126: 1505-14
Display abstract
Neurons and glial cells provide guidance cues for migrating neurons. We show here that migrating epithelial cells also contact specific neurons and glia during their pathfinding, and we describe the first gene required in the process. In wild-type Drosophila embryos, the ganglionic tracheal branch navigates a remarkably complex path along specific neural and glial substrata, switching substrata five times before reaching its ultimate target in the CNS. In adrift mutants, ganglionic branches migrate normally along the intersegmental nerve, but sporadically fail to switch to the segmental nerve and enter the CNS; they wind up meandering along the ventral epidermis instead. adrift encodes a novel nuclear protein with an evolutionarily conserved motif. The gene is required in the trachea and is expressed in the leading cells of migrating ganglionic branches where it is induced by the branchless FGF pathway. We propose that Adrift regulates expression of tracheal genes required for pathfinding on the segmental nerve, and FGF induction of adrift expression in migrating tracheal cells promotes the switch from the intersegmental to the segmental nerve.

Grebe TW, Stock JB
The histidine protein kinase superfamily.
Adv Microb Physiol. 1999; 41: 139-227
Display abstract
Signal transduction in microorganisms and plants is often mediated by His-Asp phosphorelay systems. Two conserved families of proteins are centrally involved: histidine protein kinases and phospho-aspartyl response regulators. The kinases generally function in association with sensory elements that regulate their activities in response to environmental signals. A sequence analysis with 348 histidine kinase domains reveals that this family consists of distinct subgroups. A comparative sequence analysis with 298 available receiver domain sequences of cognate response regulators demonstrates a significant correlation between kinase and regulator subfamilies. These findings suggest that different subclasses of His-Asp phosphorelay systems have evolved independently of one another.

Hoff WD et al.
Global conformational changes upon receptor stimulation in photoactive yellow protein.
Biochemistry. 1999; 38: 1009-17
Display abstract
Biological signal transduction starts with the activation of a receptor protein. Two central questions in signaling are the mechanism of activation by a stimulus and the nature and extent of the protein conformational changes involved. We report extensive evidence for the occurrence of large structural changes upon the light activation of photoactive yellow protein (PYP), a eubacterial photosensor. Absorption of a blue photon by the p-coumaric acid (pCA) chromophore in pG, the initial state of PYP, results in the formation of pB, a putative signaling state. In the presence of an adequate hydration shell, large structural changes in the protein backbone, involving both solvent accessible and core regions, were detected using Fourier transform infrared (FTIR) difference spectroscopy. A significant part (23%) of the amide groups which are buried in pG become exposed to the solvent in pB, as measured through light-induced H/D exchange, using both electrospray ionization mass spectrometry and FTIR spectroscopy. Exposure of previously buried hydrophobic sites would lead to an increase in heat capacity during pB formation and a decrease in heat capacity during pB decay. Thermodynamic studies indeed show that the heat capacity change of pB activation is -2.35 +/- 0.08 kJ/(mol/K), independent of pH from pH 2.4-7.5. A model for photoactivation of PYP is proposed, which provides a framework for a deeper understanding of receptor activation in general.

Perman B et al.
Energy transduction on the nanosecond time scale: early structural events in a xanthopsin photocycle.
Science. 1998; 279: 1946-50
Display abstract
Photoactive yellow protein (PYP) is a member of the xanthopsin family of eubacterial blue-light photoreceptors. On absorption of light, PYP enters a photocycle that ultimately transduces the energy contained in a light signal into an altered biological response. Nanosecond time-resolved x-ray crystallography was used to determine the structure of the short-lived, red-shifted, intermediate state denoted [pR], which develops within 1 nanosecond after photoelectronic excitation of the chromophore of PYP by absorption of light. The resulting structural model demonstrates that the [pR] state possesses the cis conformation of the 4-hydroxyl cinnamic thioester chromophore, and that the process of trans to cis isomerization is accompanied by the specific formation of new hydrogen bonds that replace those broken upon excitation of the chromophore. Regions of flexibility that compose the chromophore-binding pocket serve to lower the activation energy barrier between the dark state, denoted pG, and [pR], and help initiate entrance into the photocycle. Direct structural evidence is provided for the initial processes of transduction of light energy, which ultimately translate into a physiological signal.

Bell SD, Jackson SP
Transcription in Archaea.
Cold Spring Harb Symp Quant Biol. 1998; 63: 41-51

Miura N
[Transcription factor forkhead family]
Seikagaku. 1998; 70: 290-6

Keasar C, Tobi D, Elber R, Skolnick J
Coupling the folding of homologous proteins.
Proc Natl Acad Sci U S A. 1998; 95: 5880-3
Display abstract
The empirical observation that homologous proteins fold to similar structures is used to enhance the capabilities of an ab initio algorithm to predict protein conformations. A penalty function that forces homologous proteins to look alike is added to the potential and is employed in the coupled energy optimization of several homologous proteins. Significant improvement in the quality of the computed structures (as compared with the computational folding of a single protein) is demonstrated and discussed.

Takahata S et al.
Transcriptionally active heterodimer formation of an Arnt-like PAS protein, Arnt3, with HIF-1a, HLF, and clock.
Biochem Biophys Res Commun. 1998; 248: 789-94
Display abstract
We isolated a cDNA clone encoding a polypeptide of 626 amino acids containing basic helix-loop-helix (bHLH) and PAS domains from a mouse cDNA library of P19 cells. This protein, termed Arnt3, showed the highest similarity to Arnt and Arnt2 in the bHLH and PAS regions. Arnt3 mRNA was expressed in brain, skeletal muscle, 13.5-day embryos, and P19 cells treated with retinoic acid. The partner PAS proteins of Arnt3 were searched for by the two-hybrid system in yeast, and HIF-1 alpha, HLF, and Clock among various bHLH/PAS proteins were found. Gel mobility shift analysis using nuclear extracts from 293T cells cotransfected with Arnt3 and HIF-1 alpha (or HLF) expression plasmids revealed that these complexes specifically bound the hypoxia-response element (HRE). Coexpression of Arnt3 and HIF-1 alpha (or HLF) in Arnt-deficient c4 cells enhanced transcription of a reporter gene driven by the HRE sequences. We also showed that Arnt3 contained an activation domain at the C-terminal region and a repression domain between the PAS-A and PAS-B regions.

Dambly-Chaudiere C, Vervoort M
The bHLH genes in neural development.
Int J Dev Biol. 1998; 42: 269-73
Display abstract
Groups of genes sharing similar motifs may be used at different steps of a same developmental process. In this review, we discuss the significance of this phenomenon in the case of the basic Helix-Loop-Helix (bHLH) proteins that are involved at different steps of the development of the peripheral nervous system (PNS) of Drosophila.

Kort R et al.
Sequence, chromophore extraction and 3-D model of the photoactive yellow protein from Rhodobacter sphaeroides.
Biochim Biophys Acta. 1998; 1385: 1-6
Display abstract
The photoactive yellow protein (pyp) gene has been isolated from Rhodobacter sphaeroides by probing with a homologous PCR-product. A sequence analysis shows that this pyp gene encodes a 124 AA protein with 48% identity to the three known PYPs. Downstream from pyp, a number of adjacent open reading frames were identified, including a gene encoding a CoA-ligase homologue (pCL). This latter protein is proposed to be involved in PYP chromophore activation, required for attachment to the apoprotein. We have demonstrated the presence of the chromophoric group, previously identified in PYP from Ectothiorhodospira halophila as trans 4-hydroxy cinnamic acid, in phototrophically cultured R. sphaeroides cells by capillary zone electrophoresis. The basic structure of the chromophore binding pocket in PYP has been conserved, as shown by a 3D model of R. sphaeroides PYP, constructed by homology-based molecular modelling. In addition, this model shows that R. sphaeroides PYP contains a characteristic, positively charged patch.

Graves BJ, Petersen JM
Specificity within the ets family of transcription factors.
Adv Cancer Res. 1998; 75: 1-55

Yamato T, Niimura N, Go N
Molecular dynamics study of femtosecond events in photoactive yellow protein after photoexcitation of the chromophore.
Proteins. 1998; 32: 268-75
Display abstract
Molecular dynamics simulations were carried out to study what happens in a photoreceptor protein, photoactive yellow protein (PYP), immediately after the vertical transition of the chromophore from the ground to the excited state. A photon absorption simulation was performed to investigate the movement of amino acid residues upon photoexcitation. To calculate the excited state of the chromophore, SCF-CI calculation was carried out with INDO/S Hamiltonian. We observed that some amino acid residues have strong interactions with the chromophore. Most of these amino acid residues are conserved in PYPs from three different species of bacteria. This observation indicates the biological importance of these residues.

Crews ST
Control of cell lineage-specific development and transcription by bHLH-PAS proteins.
Genes Dev. 1998; 12: 607-20

Gu YZ, Moran SM, Hogenesch JB, Wartman L, Bradfield CA
Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha.
Gene Expr. 1998; 7: 205-13
Display abstract
Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS (bHLH-PAS) superfamily. In our efforts to identify new bHLH-PAS proteins, we cloned a cDNA encoding a novel alpha-class hypoxia inducible factor, HIF3alpha. The HIF3alpha open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa and is expressed in adult thymus, lung, brain, heart, and kidney. The N-terminal bHLH-PAS domain of this protein shares amino acid sequence identity with that of HIF1alpha and HIF2alpha (57% and 53% identity, respectively). The C-terminus of HIF3alpha contains a 36-amino acid sequence that shares 61% identity with the hypoxia responsive domain-1 (HRD1) of HIF1alpha. In transient transfections, this domain confers hypoxia responsiveness when linked to a heterologous transactivation domain. In vitro studies reveal that HIF3alpha dimerizes with a prototype beta-class subunit, ARNT, and that the resultant heterodimer recognizes the hypoxia responsive element (HRE) core sequence, TACGTG. Transient transfection experiments demonstrate that the HIF3alpha-ARNT interaction can occur in vivo, and that the activity of HIF3alpha is upregulated in response to cobalt chloride or low oxygen tension.

Holm L
Unification of protein families.
Curr Opin Struct Biol. 1998; 8: 372-9
Display abstract
Computational biology exploits the evolutionary connectivity between proteins and protein families to predict structural and functional properties of uncharacterized gene products. In the past year, conceptual and statistical refinements have substantially improved algorithms for the detection of remote homologues. In conjunction with the rapid growth of biological databases, the global organization of proteins into sequence families, functional families and structural families has become both pertinent and feasible.

McEvoy MM, Hausrath AC, Randolph GB, Remington SJ, Dahlquist FW
Two binding modes reveal flexibility in kinase/response regulator interactions in the bacterial chemotaxis pathway.
Proc Natl Acad Sci U S A. 1998; 95: 7333-8
Display abstract
The crystal structure at 2.0-A resolution of the complex of the Escherichia coli chemotaxis response regulator CheY and the phosphoacceptor-binding domain (P2) of the kinase CheA is presented. The binding interface involves the fourth and fifth helices and fifth beta-strand of CheY and both helices of P2. Surprisingly, the two heterodimers in the asymmetric unit have two different binding modes involving the same interface, suggesting some flexibility in the binding regions. Significant conformational changes have occurred in CheY compared with previously determined unbound structures. The active site of CheY is exposed by the binding of the kinase domain, possibly to enhance phosphotransfer from CheA to CheY. The conformational changes upon complex formation as well as the observation that there are two different binding modes suggest that the plasticity of CheY is an essential feature of response regulator function.

Genick UK, Soltis SM, Kuhn P, Canestrelli IL, Getzoff ED
Structure at 0.85 A resolution of an early protein photocycle intermediate.
Nature. 1998; 392: 206-9
Display abstract
Protein photosensors from all kingdoms of life use bound organic molecules, known as chromophores, to detect light. A specific double bond within each chromophore is isomerized by light, triggering slower changes in the protein as a whole. The initial movements of the chromophore, which can occur in femtoseconds, are tightly constrained by the surrounding protein, making it difficult to see how isomerization can occur, be recognized, and be appropriately converted into a protein-wide structural change and biological signal. Here we report how this dilemma is resolved in the photoactive yellow protein (PYP). We trapped a key early intermediate in the light cycle of PYP at temperatures below -100 degrees C, and determined its structure at better than 1 A resolution. The 4-hydroxycinnamoyl chromophore isomerizes by flipping its thioester linkage with the protein, thus avoiding collisions resulting from large-scale movement of its aromatic ring during the initial light reaction. A protein-to-chromophore hydrogen bond that is present in both the preceding dark state and the subsequent signalling state of the photosensor breaks, forcing one of the hydrogen-bonding partners into a hydrophobic pocket. The isomerized bond is distorted into a conformation resembling that in the transition state. The resultant stored energy is used to drive the PYP light cycle. These results suggest a model for phototransduction, with implications for bacteriorhodopsin, photoactive proteins, PAS domains, and signalling proteins.

van Aalten DM, Hoff WD, Findlay JB, Crielaard W, Hellingwerf KJ
Concerted motions in the photoactive yellow protein.
Protein Eng. 1998; 11: 873-9
Display abstract
Molecular dynamics simulations have been performed with the aim of identifying concerted backbone motions in the photoactive yellow protein. Application of the essential dynamics method revealed large, chromophore-linked fluctuations of the protein in the ground state, as well as in a form containing the isomerized chromophore. Various loops become more mobile upon isomerization of the chromophore, including a loop which is part of the PAS domain motif, found in light perception proteins. The hinge points identified in these fluctuations correlate with the positions of evolutionary conserved glycines. The results derived from the simulations directly correlate with available experimental data, provide a framework for understanding the dynamic behaviour of the yellow protein and give clues to subsequent steps in the signal transduction pathway.

Georgias C, Wasser M, Hinz U
A basic-helix-loop-helix protein expressed in precursors of Drosophila longitudinal visceral muscles.
Mech Dev. 1997; 69: 115-24
Display abstract
Basic-helix-loop-helix (bHLH) transcription factors are involved in the control of many developmental processes in vertebrates and invertebrates. The HLH domain mediates formation of homo- or heterodimers. We have taken advantage of these dimerisation properties to identify a novel Drosophila HLH protein using the yeast two-hybrid system. Expression of bHLH54F at the blastoderm stage is restricted to a small subpopulation of mesodermal cells near the posterior pole. During germ band retraction these cells spread along the future midgut region. Later bHLH54F-expressing cells make up the longitudinal portion of the visceral musculature. Characterisation of this expression pattern demonstrates that precursors of the outer, longitudinal muscles of the midgut are distinct in origin and morphology from precursors of the inner, circular muscles.

Dixon MJ
Twist and shout.
Nat Genet. 1997; 15: 3-4

Genick UK et al.
Structure of a protein photocycle intermediate by millisecond time-resolved crystallography.
Science. 1997; 275: 1471-5
Display abstract
The blue-light photoreceptor photoactive yellow protein (PYP) undergoes a self-contained light cycle. The atomic structure of the bleached signaling intermediate in the light cycle of PYP was determined by millisecond time-resolved, multiwavelength Laue crystallography and simultaneous optical spectroscopy. Light-induced trans-to-cis isomerization of the 4-hydroxycinnamyl chromophore and coupled protein rearrangements produce a new set of active-site hydrogen bonds. An arginine gateway opens, allowing solvent exposure and protonation of the chromophore's phenolic oxygen. Resulting changes in shape, hydrogen bonding, and electrostatic potential at the protein surface form a likely basis for signal transduction. The structural results suggest a general framework for the interpretation of protein photocycles.

Sonnenfeld M, Ward M, Nystrom G, Mosher J, Stahl S, Crews S
The Drosophila tango gene encodes a bHLH-PAS protein that is orthologous to mammalian Arnt and controls CNS midline and tracheal development.
Development. 1997; 124: 4571-82
Display abstract
The Drosophila single-minded and trachealess bHLH-PAS genes control transcription and development of the CNS midline cell lineage and tracheal tubules, respectively. We show that Single-minded and Trachealess activate transcription by forming dimers with the Drosophila Tango protein that is an orthologue of the mammalian Arnt protein. Both cell culture and in vivo studies show that a DNA enhancer element acts as a binding site for both Single-minded::Tango and Trachealess::Tango heterodimers and functions in controlling CNS midline and tracheal transcription. Isolation and analysis of tango mutants reveal CNS midline and tracheal defects, and gene dosage studies demonstrate in vivo interactions between single-minded::tango and trachealess::tango. These experiments support the existence of an evolutionarily conserved, functionally diverse bHLH-PAS protein regulatory system.

Devanathan S, Genick UK, Getzoff ED, Meyer TE, Cusanovich MA, Tollin G
Preparation and properties of a 3,4-dihydroxycinnamic acid chromophore variant of the photoactive yellow protein.
Arch Biochem Biophys. 1997; 340: 83-9
Display abstract
Native photoactive yellow protein (PYP) is reversibly bleached by laser excitation at the 446-nm wavelength maximum, during which the trans-4-hydroxycinnamic acid chromophore (covalently bound via a thioester to Cys 69) is isomerized, causing the protein to undergo a conformational change. We have reconstituted the holoprotein from recombinant apoprotein plus thiophenol thioester-activated chromophore and have also successfully attached a synthetic 3,4-dihydroxycinnamic acid chromophore and purified the resultant variant. The reconstituted recombinant protein has the same spectral and photochemical properties as the native protein. However, the absorption maximum of the protein with the dihydroxy chromophore variant is red-shifted to 458 nm, with an additional shoulder at about 342 nm. Following a laser flash, the rate constants for the first phase of bleaching in both the native and the variant proteins are too large to measure with the present apparatus. The second bleaching phase is only marginally accessible in the variant and has a rate constant (k approximately 2.3 x 10(4) s-1) at least an order of magnitude larger than that of the native PYP. In contrast, the rate constant for recovery of absorbance in the variant (k approximately 0.15 s-1) is about 40-fold smaller than for native PYP and is insensitive to pH (the native protein has a biphasic 16-fold variation in rate constant with pH). We previously observed similar changes in kinetic rate constants for protein denatured by urea or alcohols, which suggests that the dihydroxy protein is less stable than the native PYP. This was confirmed by measurement of protein unfolding in guanidine hydrochloride. We conclude from these results that the binding site is too small to accommodate the dihydroxybenzene ring of the variant chromophore without introducing strain into the protein, which is then reflected in the kinetic properties of the photocycle.

Hahn ME, Karchner SI, Shapiro MA, Perera SA
Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family.
Proc Natl Acad Sci U S A. 1997; 94: 13743-8
Display abstract
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Cederberg A et al.
Chromosome localization, sequence analysis, and expression pattern identify FKHL 18 as a novel human forkhead gene.
Genomics. 1997; 44: 344-6
Display abstract
The forkhead gene family of transcription factors belongs to the "winged helix" class of DNA-binding proteins. Today over 40 members of this gene family have been identified. Forkhead genes have been shown to be involved in embryonic development, tumorigenesis, and direction of tissue specificity of gene expression. Here we describe a new human forkhead gene called freac-10 (HGMW-approved symbol FKHL 18). A combination of fluorescence in situ hybridization and somatic cell hybrids localizes freac-10 to the chromosomal region of 20q11.1-q11.2. Hybridization to a panel consisting of RNA derived from 50 different tissues shows that freac-10 is transcribed predominantly in the aorta, thus having a unique expression pattern compared with other forkhead genes. Sequence comparison reveals a striking similarity, over the conserved DNA binding region, to a murine forkhead gene-fkh-3. We propose, based on sequence differences in the N- and C-terminal regions of the forkhead domain and a clear difference in expression pattern between freac-10 and fkh-3, that freac-10 represents a novel member of this gene family.

Moffett P, Reece M, Pelletier J
The murine Sim-2 gene product inhibits transcription by active repression and functional interference.
Mol Cell Biol. 1997; 17: 4933-47
Display abstract
The Drosophila single-minded (Dsim) gene encodes a master regulatory protein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene products possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (designated a conserved region among PER, ARNT [aryl hydrocarbon receptor nuclear translocator] and SIM) protein association domains. Members of this family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as xenobiotics and hypoxia. We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH and PAS domains of Dsim. Immunoprecipitation experiments with recombinant proteins indicate that mSIM-2 associates with the arnt gene product. In the present work, by using fine-structure mapping we found that the HLH and PAS motifs of both proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM-2 and ARNT to heterodimerize, (ii) is dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. We also demonstrated that mSIM-2 can functionally interfere with hypoxia-inducible factor 1alpha (HIF-1alpha)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription.

Mihara K, Hisatomi O, Imamoto Y, Kataoka M, Tokunaga F
Functional expression and site-directed mutagenesis of photoactive yellow protein.
J Biochem (Tokyo). 1997; 121: 876-80
Display abstract
The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP isolated from E. halophila. The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP. The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments. These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.

Genick UK et al.
Active site mutants implicate key residues for control of color and light cycle kinetics of photoactive yellow protein.
Biochemistry. 1997; 36: 8-14
Display abstract
To understand how the protein and chromophore components of a light-sensing protein interact to create a light cycle, we performed time-resolved spectroscopy on site-directed mutants of photoactive yellow protein (PYP). Recently determined crystallographic structures of PYP in the ground and colorless I2 states allowed us to design mutants and to study their photosensing properties at the atomic level. We developed a system for rapid mutagenesis and heterologous bacterial expression for PYP apoprotein and generated holoprotein through formation of a covalent thioester linkage with the p-hydroxycinnamic acid chromophore as found in the native protein. Glu46, replaced by Gln, is buried in the active site and hydrogen bonds to the chromophore's phenolate oxygen in the ground state. The Glu46Gln mutation shifted the ground state absorption maximum from 446 to 462 nm, indicating that the color of PYP can be fine-tuned by the alteration of hydrogen bonds. Arg52, which separates the active site from solvent in the ground state, was substituted by Ala. The smaller red shift (to 452 nm) of the Arg52Ala mutant suggests that electrostatic interactions with Arg52 are not important for charge stabilization on the chromophore. Both mutations cause interesting changes in light cycle kinetics. The most dramatic effect is a 700-fold increase in the rate of recovery to the ground state of Glu46Gln PYP in response to a change in pH from pH 5 to 10 (pKa = 8). Prompted by this large effect, we conducted a careful reexamination of pH effects on the wild-type PYP light cycle. The rate of color loss decreased about 3-fold with increasing pH from pH 5 to 10. The rate of recovery to the colored ground state showed a bell-shaped pH dependence, controlled by two pKa values (6.4 and 9.4). The maximum recovery rate at pH 7.9 is about 16 times faster than at pH 5. The effect of pH on Arg52Ala is like that on wild type except for faster loss of color and slower recovery. These kinetic effects of the mutations and the changes with pH demonstrate that both phases in PYP's light cycle are actively controlled by the protein component.

Imamoto Y, Tokunaga F
[Structure and photochemical reactions of photoactive yellow protein]
Tanpakushitsu Kakusan Koso. 1997; 42: 129-35

Kay SA
As time PASses: the first mammalian clock gene.
Science. 1997; 276: 1093-1093

Imamoto Y et al.
Evidence for proton transfer from Glu-46 to the chromophore during the photocycle of photoactive yellow protein.
J Biol Chem. 1997; 272: 12905-8
Display abstract
Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at -40 degrees C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995) Biochemistry 34, 12669-12672) were observed at 1482, 1437, and 1163 cm-1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm-1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm-1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 --> Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM.

Perrin-Schmitt F, Bolcato-Bellemin AL, Bourgeois P, Stoetzel C, Danse JM
The locations of the H-twist and H-dermo-1 genes are distinct on the human genome.
Biochim Biophys Acta. 1997; 1360: 1-2

Sogawa K, Fujii-Kuriyama Y
[Structure and function of PAS proteins: Ah receptor, Arnt, and others]
Tanpakushitsu Kakusan Koso. 1996; 41: 1288-96

Gradwohl G, Fode C, Guillemot F
Restricted expression of a novel murine atonal-related bHLH protein in undifferentiated neural precursors.
Dev Biol. 1996; 180: 227-41
Display abstract
Tissue-specific bHLH proteins play important roles in the specification and differentiation of neural cell lineages in invertebrate and vertebrate organisms. Two groups of bHLH proteins, atonal and achaete-scute, have proneural activities in Drosophila, and the mouse achaete-scute homolog MASH1 is required for the differentiation of several neural lineages. In a screen for proteins interacting with MASH1, we have isolated a novel bHLH protein related to atonal, named MATH4A, which is broadly expressed in neural precursor cells in the mouse embryonic CNS and PNS. Interaction assays in yeast and in vitro demonstrate that MATH4A interacts efficiently with both MASH1 and the ubiquitous bHLH protein E12. MATH4A-E12 heterodimers, but not MATH4A-MASH1, bind to a consensus E-box sequence. Math4A expression is restricted to undifferentiated neural precursors and is complementary to that of Mash1 in most regions of the nervous system. In particular, Math4A is transcribed at high levels in the cerebral cortex, dorsal thalamus, and epibranchial placodes, which present little or no Mash1 expression. However, expression of the two genes shows limited overlap in certain CNS regions (retina, preoptic area of the hypothalamus, midbrain, hindbrain). Its structure and expression pattern suggest that MATH4A may regulate an early step of neural development, either as a partner of ubiquitous bHLH proteins or associated with other neural-specific bHLH proteins.

Taylor MV
A myogenic switch. Muscle development.
Curr Biol. 1996; 6: 924-6
Display abstract
Experiments manipulating the level of the basic helix-loop-helix transcription factor Twist in the Drosophila embryo have revealed a novel role for this protein in a 'myogenic switch' during early development.

Koh M et al.
Sequence evidence for strong conservation of the photoactive yellow proteins from the halophilic phototrophic bacteria Chromatium salexigens and Rhodospirillum salexigens.
Biochemistry. 1996; 35: 2526-34
Display abstract
The photoactive yellow proteins (PYP) have been found to date only in three species of halophilic purple phototrophic bacteria. They have photochemical activity remarkably similar to that of the bacteria rhodopsins. In contrast to rhodopsins, however, the PYPs are small water-soluble proteins. We now report the complete amino acid sequences of Rhodospirillum salexigens and Chromatium salexigens PYP which allow comparison with the known sequence and three-dimensional structure of the prototypic protein from Ectothiorhodospira halophila. Although isolated from three different families of bacteria, the PYP sequences are 70-76% identical. All three contain 125 amino acid residues, and no insertions or deletions are necessary for alignment. This is a remarkable result when it is considered that electron transfer proteins from these purple bacterial species are only 25-40% identical and that insertions and deletions are needed for their proper alignment. It thus appears that PYP has the same important function in each of the purple bacteria and that most of the amino acid residues are necessary to maintain structure and function. By most standards, PYP would be called a "slowly evolving protein". R. salexigens PYP is uniquely degraded by proteolysis at low ionic strength, probably as a consequence of unfolding due to electrostatic repulsion of the excess negative charge. Therefore it may also be classified as a "halophilic protein".

Nambu JR, Chen W, Hu S, Crews ST
The Drosophila melanogaster similar bHLH-PAS gene encodes a protein related to human hypoxia-inducible factor 1 alpha and Drosophila single-minded.
Gene. 1996; 172: 249-54
Display abstract
The Drosophila melanogaster (Dm) similar (sima) gene was isolated using a low-stringency hybridization screen employing a Dm single-minded gene basic helix-loop-helix (bHLH) DNA probe. sima is a member of the bHLH-PAS gene family and the conceptual protein shares a number of structural features, including a bHLH domain, PAS domain, and homopolymeric amino acid stretches. Sima is most closely related to the human hypoxia-inducible factor 1 alpha bHLH-PAS protein. In situ hybridization experiments reveal that sima is transcribed in most or all cells throughout embryogenesis. It has been cytologically mapped to position 99D on the third chromosome, and is not closely linked to other known bHLH-PAS genes.

Theopold U, Ekengren S, Hultmark D
HLH106, a Drosophila transcription factor with similarity to the vertebrate sterol responsive element binding protein.
Proc Natl Acad Sci U S A. 1996; 93: 1195-9
Display abstract
We cloned a Drosophila homolog to the sterol responsive element binding proteins (SREBPs). In vertebrates, the SREBPs are regulated by a mechanism that involves cleavage of the protein that normally residues in the cellular membranes and translocation of the released transcription factor into the nucleus. Regulation of the Drosophila factor HLH106 apparently follows the same mechanism, and we find the full-length gene product in the membrane fraction and a shorter cross-reacting form in the nuclear fraction. This nuclear form, which may correspond to proteolytically activated HLH106, is abundant in the blood cell line mbn-2. The general domain structure of HLH106 is very similar to that in SREBP. HLH106 is expressed throughout development, and it is present at high levels in Drosophila cell lines. In contrast to the rat homolog, HLH106 transcripts are not more abundant in adipose tissue than in other tissues.

Xie A, Hoff WD, Kroon AR, Hellingwerf KJ
Glu46 donates a proton to the 4-hydroxycinnamate anion chromophore during the photocycle of photoactive yellow protein.
Biochemistry. 1996; 35: 14671-8
Display abstract
Photoactive yellow protein (PYP) is a photoreceptor containing a unique 4-hydroxycinnamic acid (pCA) chromophore. The trans to cis photoisomerization of this chromophore activates a photocycle involving first a short-lived red-shifted intermediate (pR), then a long-lived blue-shifted intermediate (pB), and finally recovery of the original receptor state (pG). The pCA chromophore is deprotonated in pG and protonated in pB, but the proton donor for this process has not yet been identified. Here we report the first FTIR spectroscopic data on pG, pR, and pB. The IR difference signals in the carbonyl stretching region of COOH groups (1700-1800 cm-1) reveal that a buried carboxylic group close to the chromophore (i) is protonated in pG, (ii) develops a stronger hydrogen bonding in pR, and (iii) becomes deprotonated in pB. These signals are unambiguously assigned to Glu46, on the basis of the IR data and the 1.4 A X-ray structure of PYP [Borgstahl et al. (1995) Biochemistry 34, 6278-6287]. Our data demonstrate that in pR Glu46 remains in hydrogen bonding contact with the negatively charged phenolic oxygen of pCA after chromophore photoisomerization. This strongly implies that the chromophore is isomerized to the 7-cis 9-s-trans conformation in pR, resulting from co-isomerization of both the C7 = C8 and C9-C10 bonds. In the pR to pB transition, Glu46 becomes deprotonated, concomitant with chromophore protonation. Therefore, we conclude that Glu46 functions as the proton donor for the protonation of pCA during the PYP photocycle. We propose a molecular mechanism in which intramolecular proton transfer in PYP leads to global protein conformational changes involved in signal transduction.

Swanson HI, Chan WK, Bradfield CA
DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins.
J Biol Chem. 1995; 270: 26292-302
Display abstract
The Ah receptor (AHR), the Ah receptor nuclear translocator protein (ARNT), and single-minded protein (SIM) are members of the basic helix-loop-helix-PAS (bHLH-PAS) family of regulatory proteins. In this study, we examine the DNA half-site recognition and pairing rules for these proteins using oligonucleotide selection-amplification and coprecipitation protocols. Oligonucleotide selection-amplification revealed that a variety of bHLH-PAS protein combinations could interact, with each generating a unique DNA binding specificity. To validate the selection-amplification protocol, we demonstrated the preference of the AHR.ARNT complex for the sequence commonly found in dioxin-responsive enhancers in vivo (TNGCGTG). We then demonstrated that the ARNT protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence, CACGTG. Further examination indicated that ARNT may have a relaxed partner specificity, since it was also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG. Coprecipitation experiments using various PAS proteins and ARNT were consistent with the idea that the ARNT protein has a broad range of interactions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions. Comparison of this in vitro data with sites known to be bound in vivo suggests that the high affinity half-site recognition sequences for the AHR, SIM, and ARNT are T(C/T)GC, GT(G/A)C (5'-half-sites), and GTG (3'-half-sites), respectively.

Ng K, Getzoff ED, Moffat K
Optical studies of a bacterial photoreceptor protein, photoactive yellow protein, in single crystals.
Biochemistry. 1995; 34: 879-90
Display abstract
Photoactive yellow protein (PYP), isolated from Ectothiorhodospira halophila, is a water soluble, 14 kDa photoreceptor protein with a fully reversible photocycle resembling that of sensory rhodopsin II. We have established the presence of photoactivity in PYP crystals and defined the relaxation kinetics of spectroscopically distinguishable species in quantitative terms. The PYP crystal has a bright yellow color and displays pronounced anisotropic absorption properties. Linear dichroism measurements show that the transition moment of the PYP chromophore makes an angle of 73 degrees (or 107 degrees) with respect to the six-fold crystallographic symmetry axis. The crystal absorbance can be bleached reversibly as indicated by absorption changes. A bleached photostationary state in the crystal can be established via CW laser illumination, and the extent of crystal bleaching is found to be clearly dependent on excitation laser wavelength, intensity and illumination time. These results provide the information for designing time-resolved crystallography experiments in which a minimum perturbation is applied to the PYP crystals. Global exponential fitting shows that the relaxation from the photostationary state in the crystal is biphasic at -4 degrees C; a slower component of 1.4 +/- 0.2 s-1 accounts for 60% of the absorbance change and a faster component of 5.2 +/- 0.9 s-1 for the other 40%. As a control, we found that the kinetics for the same relaxation in solution are well described by one exponential and agree quantitatively with previous studies. The two rate constants observed in the crystal show similar temperature dependences, with activation energies for the slow and fast components of 11.7 +/- 1.2 and 5.5 +/- 2.3 kcal/mol, respectively. However, the amplitudes associated with the two exponents show different and opposite temperature dependence. Our results show that the solution kinetic model is not directly applicable to crystals. A kinetic model consistent with the optical data is important to extract the underlying structural intermediates from the time-resolved X-ray diffraction data obtained in parallel with the optical data described here. We propose an alternative model for the photocycle in the crystal which contains an additional bleached intermediate in parallel with the last long-lived intermediate in the solution model.

Imamoto Y, Ito T, Kataoka M, Tokunaga F
Reconstitution photoactive yellow protein from apoprotein and p-coumaric acid derivatives.
FEBS Lett. 1995; 374: 157-60
Display abstract
We report reconstitution of photoactive yellow protein (PYP) from apoPYP and p-coumaric acid derivatives. The addition of p-coumaric acid to the apoPYP sample did not result in the recovery of PYP. In contrast, yellow products were obtained by the addition of p-coumaryl thiophenyl ester or p-coumaric anhydride to the apoPYP sample, the absorption spectra of which were indistinguishable from the spectrum of intact PYP. Our findings provide strong evidence that PYP has the p-coumaryl chromophore. This reconstitution technique opens the way for further biophysical studies of PYP using artificial chromophore analogs.

McGuire J, Coumailleau P, Whitelaw ML, Gustafsson JA, Poellinger L
The basic helix-loop-helix/PAS factor Sim is associated with hsp90. Implications for regulation by interaction with partner factors.
J Biol Chem. 1995; 270: 31353-7
Display abstract
Sim is a Drosophila developmental basic helix-loop-helix (bHLH) transcription factor containing a Per-Arnt-Sim (PAS) region of homology. Here we demonstrate that Sim, in analogy to the structurally related bHLH/PAS dioxin receptor, was stably associated with the molecular chaperone hsp90. In the case of the dioxin receptor, release of hsp90 and derepression of receptor function appear to be regulated by ligand binding and dimerization with Arnt, a non-hsp90-associated bHLH/PAS factor. Dimerization with Arnt very efficiently disrupted Sim-hsp90 interaction, a process that required both the bHLH and PAS dimerization motifs of Arnt. Moreover, hsp90 was also released upon dimerization of Sim with the Drosophila PAS factor Per, whereas the hsp90-associated dioxin receptor failed to interact with Sim. These results indicate that hsp90 may play a role in conditional regulation of Sim function, and that Per and possibly bHLH/PAS partner factors may activate Sim by inducing release of hsp90 during the dimerization process.

van Brederode ME, Gensch T, Hoff WD, Hellingwerf KJ, Braslavsky SE
Photoinduced volume change and energy storage associated with the early transformations of the photoactive yellow protein from Ectothiorhodospira halophila.
Biophys J. 1995; 68: 1101-9
Display abstract
The photocycle of the photoactive yellow protein (PYP) isolated from Ectothiorhodospira halophila was analyzed by flash photolysis with absorption detection at low excitation photon densities and by temperature-dependent laser-induced optoacoustic spectroscopy (LIOAS). The quantum yield for the bleaching recovery of PYP, assumed to be identical to that for the phototransformation of PYP (pG), to the red-shifted intermediate, pR, was phi R = 0.35 +/- 0.05, much lower than the value of 0.64 reported in the literature. With this value and the LIOAS data, an energy content for pR of 120 kJ/mol was obtained, approximately 50% lower than for excited pG. Concomitant with the photochemical process, a volume contraction of 14 ml/photoconverted mol was observed, comparable with the contraction (11 ml/mol) determined for the bacteriorhodopsin monomer. The contraction in both cases is interpreted to arise from a protein reorganization around a phototransformed chromophore with a dipole moment different from that of the initial state. The deviations from linearity of the LIOAS data at photon densities > 0.3 photons per molecule are explained by absorption by pG and pR during the laser pulse duration (i.e., a four-level system, pG, pR, and their respective excited states). The data can be fitted either by a simple saturation process or by a photochromic equilibrium between pG and pR, similar to that established between the parent chromoprotein and the first intermediate(s) in other biological photoreceptors. This nonlinearity has important consequences for the interpretation of the data obtained from in vitro studies with powerful lasers.

Kim M, Mathies RA, Hoff WD, Hellingwerf KJ
Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated.
Biochemistry. 1995; 34: 12669-72
Display abstract
Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy.

Wulbeck C, Fromental-Ramain C, Campos-Ortega JA
The HLH domain of a zebrafish HE12 homologue can partially substitute for functions of the HLH domain of Drosophila DAUGHTERLESS.
Mech Dev. 1994; 46: 73-85
Display abstract
We have identified a zebrafish homologue of the human E12 protein, which we have called ZFE12. It shows a high degree of identity to HE12 throughout its entire sequence, particularly in the bHLH domain (93%); amino acid sequence identity of the bHLH domain of ZFE12 to Drosophila DAUGHTERLESS is also very high (75%). During early embryogenesis, expression of ZfE12 is widespread. Following gastrulation, ZfE12 transcripts can be detected in all embryonic tissues with the exception of the notochord, although zones with relatively higher densities of transcripts are present in the developing brain and in the somites. To assay biological activities associated with the ZFE12 protein, two P-element constructs were made, each carrying a Drosophila daughterless gene that had been modified by replacing either the HLH domain or the entire C-terminus including the bHLH domain, by the equivalent domains of ZfE12. These constructs were used to transform flies and tested for their ability to rescue the daughterless mutant phenotype. Complete rescue of the neural phenotype and of the embryonic lethality was obtained. However, the daughterless function could not be completely restored. Although fertile flies transheterozygous for a hypomorphic and an amorphic mutation and carrying the construct encoding the zebrafish HLH domain emerged, they lacked various types of sensory organs on head and thorax and showed slight wing defects. Mutants carrying the second construct occasionally reached pupal stages but died subsequently. These data demonstrate that the HLH domain of ZFE12 can carry out most, but not all, functions performed by the corresponding region of the DAUGHTERLESS protein in Drosophila.

Hoff WD et al.
The photoactive yellow protein from Ectothiorhodospira halophila as studied with a highly specific polyclonal antiserum: (intra)cellular localization, regulation of expression, and taxonomic distribution of cross-reacting proteins.
J Bacteriol. 1994; 176: 3920-7
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A rabbit antiserum was raised against the photoactive yellow protein (PYP) from Ectothiorhodospira halophila and purified by adsorption experiments to obtain a highly specific polyclonal antiserum. This antiserum was used to obtain the following results. (i) In E. halophila, PYP can be isolated from the fraction of soluble proteins. In the intact cell, however, PYP appeared to be associated with (intra)cytoplasmic membranes, as was concluded from analysis of immunogold-labelled thin sections of the organism. (ii) The regulation of expression of PYP was studied by using dot blot assays, Western blotting (immunoblotting), and rocket immunoelectrophoresis. Under all conditions investigated (light color, salt concentration, and growth phase), PYP was expressed constitutively in E. halophila. However, when Rhodospirillum salexigens was grown aerobically, the expression of PYP was suppressed. (iii) A large number of prokaryotic microorganisms contained a single protein, with an apparent size of approximately 15 kDa, that cross-reacted with the antiserum. Among the positively reacting organisms were both phototrophic and chemotrophic, as well as motile and nonmotile, organisms. After separation of cellular proteins into a membrane fraction and soluble proteins, it was established that organisms adapted to growth at higher salt concentrations tended to have the cross-reacting protein in the soluble fraction. In the cases of R. salexigens and Chromatium salexigens, we have shown that the cross-reacting protein involved is strongly homologous to PYP from E. halophila.

Hoff WD et al.
Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.
Biophys J. 1994; 67: 1691-705
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The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.

Ohsako S, Hyer J, Panganiban G, Oliver I, Caudy M
Hairy function as a DNA-binding helix-loop-helix repressor of Drosophila sensory organ formation.
Genes Dev. 1994; 8: 2743-55
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Sensory organ formation in Drosophila is activated by proneural genes that encode basic-helix-loop-helix (bHLH) transcription factors. These genes are antagonized by hairy and other proline-bHLH proteins. hairy has not been shown to bind to DNA and has been proposed to form inactive heterodimers with proneural activator proteins. Here, we show that hairy does bind to DNA and has novel DNA-binding activity: hairy prefers a noncanonical site, CACGCG, although it also binds to related sites. Mutation of a single CACGCG site in the achaete (ac) proneural gene blocks hairy-mediated repression of ac transcription in cultured Drosophila cells. Moreover, the same CACGCG mutation in an ac minigene transformed into Drosophila creates ectopic sensory hair organs like those seen in hairy mutants. Together these results indicate that hairy represses sensory organ formation by directly repressing transcription of the ac proneural gene.

Baca M et al.
Complete chemical structure of photoactive yellow protein: novel thioester-linked 4-hydroxycinnamyl chromophore and photocycle chemistry.
Biochemistry. 1994; 33: 14369-77
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The unique ability of photoactive proteins to capture and use energy from a photon of light depends on the chromophore, its linkage to the protein, and the surrounding protein environment. To understand the molecular mechanisms by which a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein (PYP), a 14-kDa water-soluble photoreceptor from Ectothiorhodospira halophila with a photocycle similar to that of sensory rhodopsin. Here, we report the cloning and sequencing of the pyp gene and the chemical identification of both the chromophore and its covalent linkage to the protein. Elemental composition data from high-resolution mass spectrometry of a proteolytically derived chromopeptide, pH titrations and UV-visible spectroscopy of the protein-bound and chemically released chromophore, and fragmentation mass spectrometry of the liberated chromophore amide were combined with results from the 1.4-A-resolution protein crystal structure to identify the chromophore in PYP as a 4-hydroxycinnamyl group covalently bound to the sole cysteine residue via a thioester linkage. While 4-hydroxycinnamate is a metabolic product of the phenylpropanoid pathway and a key molecule in plant stress response, this is the first report of covalent modification of a protein by this group. In the dark (yellow) state of PYP, the protein stabilizes the chromophore as the deprotonated phenolate anion. By combining our biochemical characterization of the chromophore with other published observations, we propose a chemical basis for the photocycle: following the initial absorption of a photon, the photocycle of PYP involves protonation of the chromophore to a neutral phenol form corresponding to the observed photobleached intermediate.

Meyer TE, Cusanovich MA, Tollin G
Transient proton uptake and release is associated with the photocycle of the photoactive yellow protein from the purple phototrophic bacterium Ectothiorhodospira halophila.
Arch Biochem Biophys. 1993; 306: 515-7
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Upon excitation by a laser flash at 445 nm, the photoactive yellow protein (PYP) undergoes a bleach and red-shift occurring in less than 10 ns, undergoes a further bleach in approximately 200 microseconds, and then recolors in approximately 200 ms. A conformational change occurs during photobleaching which exposes a hydrophobic site. We have now shown that this photocycle also involves a net uptake of one proton during formation of the fully bleached second intermediate, followed by an equivalent proton release upon return of PYP to the ground state. Proton uptake lags slightly behind PYP bleaching and is first-order, indicating that the protein conformational change occurs in two steps. The results suggest that a basic residue which is normally buried in the protein interior is transiently exposed to solvent during the PYP photocycle and, as a consequence, undergoes a change in pK. On the basis of the crystal structure of PYP, we propose that this basic residue is lysine 111.

Van Beeumen JJ et al.
Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila, with evidence for the mass and the binding site of the chromophore.
Protein Sci. 1993; 2: 1114-25
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The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.

Kahn D
Shuffling of an oxygen sensor haem domain.
Mol Microbiol. 1993; 8: 786-7

Huang ZJ, Edery I, Rosbash M
PAS is a dimerization domain common to Drosophila period and several transcription factors.
Nature. 1993; 364: 259-62
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Mutations in the period gene product (PER) can shorten or lengthen the circadian rhythms of Drosophila melanogaster, but its biochemical activity has not been established. PER contains a motif of approximately 270 amino acids whose function is unknown (termed PAS) and which is also present in three transcription factors of the basic-helix-loop-helix (bHLH) type, in the D. melanogaster single-minded gene product (SIM), and in both subunits of the mammalian dioxin receptor complex. We show here that the PER PAS functions in vitro as a novel protein dimerization motif and that it can mediate associations between different members of the PAS protein family. The dimerization efficiency is decreased by several missense mutations in the PAS domain, including the original perL mutation, which lengthens circadian periods from 24 h to 29 h (ref. 1). The results indicate that the PAS domain may function as a dimerization domain in both SIM and the dioxin receptor complex, and that PER may regulate circadian gene transcription partly by interacting with the PAS domain of bHLH--PAS-containing transcription factors.