NORMAL GENOMIC

• Philip AF, Nome RA, Papadantonakis GA, Scherer NF, Hoff WD
• Spectral tuning in photoactive yellow protein by modulation of the shapeof the excited state energy surface.
• Proc Natl Acad Sci U S A. 2010; 107: 5821-6
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• Protein-chromophore interactions in photoreceptors often shift thechromophore absorbance maximum to a biologically relevant spectral region.A fundamental question regarding such spectral tuning effects is how theelectronic ground state S(0) and excited state S(1) are modified by theprotein. It is widely assumed that changes in energy gap between S(0) andS(1) are the main factor in biological spectral tuning. We report agenerally applicable approach to determine if a specific residue modulatesthe energy gap, or if it alters the equilibrium nuclear geometry or widthof the energy surfaces. This approach uses the effects that changes inthese three parameters have on the absorbance and fluorescence emissionspectra of mutants. We apply this strategy to a set of mutants ofphotoactive yellow protein (PYP) containing all 20 side chains at activesite residue 46. While the mutants exhibit significant variation in boththe position and width of their absorbance spectra, the fluorescenceemission spectra are largely unchanged. This provides strong evidenceagainst a major role for changes in energy gap in the spectral tuning ofthese mutants and reveals a change in the width of the S(1) energysurface. We determined the excited state lifetime of selected mutants andthe observed correlation between the fluorescence quantum yield andlifetime shows that the fluorescence spectra are representative of theenergy surfaces of the mutants. These results reveal that residue 46 tunesthe absorbance spectrum of PYP largely by modulating the width of the S(1)energy surface.

• Qi Y, Rao F, Luo Z, Liang ZX
• A flavin cofactor-binding PAS domain regulates c-di-GMP synthesis inAxDGC2 from Acetobacter xylinum.
• Biochemistry. 2009; 48: 10275-85
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• The cytoplasmic protein AxDGC2 regulates cellulose synthesis in theobligate aerobe Acetobacter xylinum by controlling the cellularconcentration of the cyclic dinucleotide messenger c-di-GMP. AxDGC2contains a Per-Arnt-Sim (PAS) domain and two putative catalytic domains(GGDEF and EAL) for c-di-GMP metabolism. We found that the PAS domain ofAxDGC2 binds a flavin adenine dinucleotide (FAD) cofactor noncovalently.The redox status of the FAD cofactor modulates the catalytic activity ofthe GGDEF domain for c-di-GMP synthesis, with the oxidized form exhibitinghigher catalytic activity and stronger substrate inhibition. The resultssuggest that AxDGC2 is a signaling protein that regulates the cellularc-di-GMP level in response to the change in cellular redox status oroxygen concentration. Moreover, several residues predicated to be involvedin FAD binding and signal transduction were mutated to examine the impacton redox potential and catalytic activity. Despite the minor perturbationof redox potential and unexpected modification of FAD in one of themutants, none of the single mutations was able to completely disrupt thetransmission of the signal to the GGDEF domain, indicating that the changein the FAD redox state can still trigger structural changes in the PASdomain probably by using substituted hydrogen-bonded water networks.Meanwhile, although the EAL domain of AxDGC2 was found to be catalyticallyinactive toward c-di-GMP, it was capable of hydrolyzing somephosphodiester bond-containing nonphysiological substrates. Together withthe previously reported oxygen-dependent activity of the homologousAxPDEA1, the results provided new insight into relationships among oxygenlevel, c-di-GMP concentration, and cellulose synthesis in A. xylinum.

• Lyons TW, Reinhard CT, Scott C
• Redox redux.
• Geobiology. 2009; 7: 489-94

• Vinogradov SN, Moens L
• Diversity of globin function: enzymatic, transport, storage, and sensing.
• J Biol Chem. 2008; 283: 8773-7

• Philip AF, Eisenman KT, Papadantonakis GA, Hoff WD
• Functional tuning of photoactive yellow protein by active site residue 46.
• Biochemistry. 2008; 47: 13800-10
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• Protein-ligand interactions alter the properties of active site groups toachieve specific biological functions. The active site of photoactiveyellow protein (PYP) provides a model system for studying such functionaltuning. PYP is a small bacterial photoreceptor with photochemistry basedon its p-coumaric acid (pCA) chromophore. The absorbance maximum and pK(a)of the pCA in the active site of native PYP are shifted from 400 nm and8.8 in water to 446 nm and 2.8 in the native protein milieu, respectively,by protein-ligand interactions. We report high-throughput microscalemethods for the purification and spectroscopic investigation of PYP anduse these to examine the role of active site residue Glu46 in PYP, whichis hydrogen bonded to the pCA anion. The functional and structuralattributes of the 19 substitution mutants of PYP at critical active siteposition 46 vary widely, with absorbance maxima from 441 to 478 nm, pCAfluorescence quantum yields from 0.19 to 1.4%, pCA pK(a) values from 3.0to 9.0, and protein folding stabilities from 6.5 to 12.9 kcal/mol. Thekinetics of the last photocycle transition vary by more than 4 orders ofmagnitude and are often strongly biphasic. Only E46Q PYP exhibits agreatly accelerated photocycling rate. All substitutions yield a folded,photoactive PYP, illustrating the robustness of protein structure andfunction. Correlations between side chain and mutant properties establishthe importance of residue 46 in tuning the function of PYP and thesignificance of the strength of its hydrogen bond to the pCA. Native PYPexhibits the lowest values for pCA fluorescence quantum yield and pK(a),indicating their functional relevance. These results demonstrate the valueof quantitative high-throughput biophysical studies of proteins.

• Schmoll M, Franchi L, Kubicek CP
• Envoy, a PAS/LOV domain protein of Hypocrea jecorina (Anamorph Trichodermareesei), modulates cellulase gene transcription in response to light.
• Eukaryot Cell. 2005; 4: 1998-2007
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• Envoy, a PAS/LOV domain protein with similarity to the Neurospora lightregulator Vivid, which has been cloned due to its lack of expression in acellulase-negative mutant, links cellulase induction by cellulose to lightsignaling in Hypocrea jecorina. Despite their similarity, env1 could notcompensate for the lack of vvd function. Besides the effect of light onsporulation, we observed a reduced growth rate in constant light. Anenv1(PAS-) mutant of H. jecorina grows significantly slower in thepresence of light but remains unaffected in darkness compared to thewild-type strain QM9414. env1 rapidly responds to a light pulse, with thisresponse being different upon growth on glucose or glycerol, and itencodes a regulator essential for H. jecorina light tolerance. Theinduction of cellulase transcription in H. jecorina by cellulose isenhanced by light in the wild-type strain QM9414 compared to that inconstant darkness, whereas a delayed induction in light and only atransient up-regulation in constant darkness of cbh1 was observed in theenv1(PAS-) mutant. However, light does not lead to cellulase expression inthe absence of an inducer. We conclude that Envoy connects the lightresponse to carbon source signaling and thus that light must be consideredan additional external factor influencing gene expression analysis in thisfungus.

• Pohlschroder M, Dilks K, Hand NJ, Wesley Rose R
• Translocation of proteins across archaeal cytoplasmic membranes.
• FEMS Microbiol Rev. 2004; 28: 3-24
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• All cells need to transport proteins across hydrophobic membranes. Severalmechanisms have evolved to facilitate this transport, including: (i) theuniversally-conserved Sec system, which transports proteins in an unfoldedconformation and is thought to be the major translocation pathway in mostorganisms and (ii) the Tat system, which transports proteins that havealready obtained some degree of tertiary structure. Here, we present thecurrent understanding of these processes in the domain Archaea, and howthey compare to the corresponding pathways in bacteria and eukaryotes.

• Levin I, Giladi M, Altman-Price N, Ortenberg R, Mevarech M
• An alternative pathway for reduced folate biosynthesis in bacteria andhalophilic archaea.
• Mol Microbiol. 2004; 54: 1307-18
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• Whereas tetrahydrofolate is an essential cofactor in all bacteria, thegene that encodes the enzyme dihydrofolate reductase (DHFR) could not beidentified in many of the bacteria whose genomes have been entirelysequenced. In this communication we show that the halophilic archaeaHalobacterium salinarum and Haloarcula marismortui contain genes codingfor proteins with an N-terminal domain homologous to dihydrofolatesynthase (FolC) and a C-terminal domain homologous to dihydropteroatesynthase (FolP). These genes are able to complement a Haloferax volcaniimutant that lacks DHFR. We also show that the Helicobacter pyloridihydropteroate synthase can complement an Escherichia coli mutant thatlacks DHFR. Activity resides in an N-terminal segment that is homologousto the polypeptide linker that connects the dihydrofolate synthase anddihydropteroate synthase domains in the haloarchaeal enzymes. The purifiedrecombinant H. pylori dihydropteroate synthase was found to be aflavoprotein.

• Davidson AL, Chen J
• ATP-binding cassette transporters in bacteria.
• Annu Rev Biochem. 2004; 73: 241-68
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• ATP-binding cassette (ABC) transporters couple ATP hydrolysis to theuptake and efflux of solutes across the cell membrane in bacteria andeukaryotic cells. In bacteria, these transporters are important virulencefactors because they play roles in nutrient uptake and in secretion oftoxins and antimicrobial agents. In humans, many diseases, such as cysticfibrosis, hyperinsulinemia, and macular dystrophy, are traced to defectsin ABC transporters. Recent advances in structural determination andfunctional analysis of bacterial ABC transporters, reviewed herein, havegreatly increased our understanding of the molecular mechanism oftransport in this transport superfamily.

• Anderson S, Crosson S, Moffat K
• Short hydrogen bonds in photoactive yellow protein.
• Acta Crystallogr D Biol Crystallogr. 2004; 60: 1008-16
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• Eight high-resolution crystal structures of the ground state ofphotoactive yellow protein (PYP) solved under a variety of conditionsreveal that its chromophore is stabilized by two unusually short hydrogenbonds. Both Tyr42 Oeta and Glu46 Oepsilon are separated from thechromophore phenolate oxygen by less than the sum of their atomic van derWaals radii, 2.6 angstroms. This is characteristic of strong hydrogenbonding, in which hydrogen bonds acquire significant covalent character.The hydrogen bond from the protonated Glu46 to the negatively chargedphenolate oxygen is 2.58 +/- 0.01 angstroms in length, while that fromTyr42 is considerably shorter, 2.49 +/- 0.01 angstroms. The E46Q mutantwas solved to 0.95 angstroms resolution; the isosteric mutation increasedthe length of the hydrogen bond from Glx46 to the chromophore by 0.29 +/-0.01 angstroms to that of an average hydrogen bond, 2.88 +/- 0.01angstroms. The very short hydrogen bond from Tyr42 explains why mutatingthis residue has such a severe effect on the ground-state structure andPYP photocycle. The effect of isosteric mutations on the photocycle can belargely explained by the alterations to the length and strength of thesehydrogen bonds.

• Winnen B, Hvorup RN, Saier MH Jr
• The tripartite tricarboxylate transporter (TTT) family.
• Res Microbiol. 2003; 154: 457-65
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• Extracytoplasmic solute binding receptors are constituents of primary andsecondary active transport systems. Previous studies have shown that theconstituents of two such families (ABC and TRAP-T) occur in bacteria andarchaea and have undergone minimal shuffling of constituents betweensystems during evolutionary history. We here show that a third family ofbinding receptor-dependent transporters, the tripartite tricarboxylatetransporter (TTT) family, the prototype of which is the TctABC system ofSalmonella typhimurium, occurs in many bacteria but not in archaea oreukaryotes. Phylogenetic analyses suggest that these systems have evolvedfrom a primordial tripartite system with only two out of 39 possibleexamples of shuffling of constituents between systems. The occurrence ofTctA homologues in many bacteria and archaea that apparently lackcorresponding TctB and TctC homologues suggests that the appearance oftripartite systems was a relatively recent evolutionary invention thatoccurred after the divergence of archaea and eukaryotes from bacteria.

• Dilks K, Rose RW, Hartmann E, Pohlschroder M
• Prokaryotic utilization of the twin-arginine translocation pathway: agenomic survey.
• J Bacteriol. 2003; 185: 1478-83
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• The twin-arginine translocation (Tat) pathway, which has been identifiedin plant chloroplasts and prokaryotes, allows for the secretion of foldedproteins. However, the extent to which this pathway is used among theprokaryotes is not known. By using a genomic approach, a comprehensivelist of putative Tat substrates for 84 diverse prokaryotes wasestablished. Strikingly, the results indicate that the Tat pathway isutilized to highly varying extents. Furthermore, while many prokaryotesuse this pathway predominantly for the secretion of redox proteins,analyses of the predicted substrates suggest that certain bacteria andarchaea secrete mainly nonredox proteins via the Tat pathway. While nocorrelation was observed between the number of Tat machinery componentsencoded by an organism and the number of predicted Tat substrates, it wasnoted that the composition of this machinery was specific to phylogenetictaxa.

• van Aalten DM, Haker A, Hendriks J, Hellingwerf KJ, Joshua-Tor L, Crielaard W
• Engineering photocycle dynamics. Crystal structures and kinetics of threephotoactive yellow protein hinge-bending mutants.
• J Biol Chem. 2002; 277: 6463-8
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• Crystallographic and spectroscopic analyses of three hinge-bending mutantsof the photoactive yellow protein are described. Previous studies haveidentified Gly(47) and Gly(51) as possible hinge points in the structureof the protein, allowing backbone segments around the chromophore toundergo large concerted motions. We have designed, crystallized, andsolved the structures of three mutants: G47S, G51S, and G47S/G51S. Theprotein dynamics of these mutants are significantly affected. Transitionsin the photocycle, measured with laser induced transient absorptionspectroscopy, show rates up to 6-fold different from the wild type proteinand show an additive effect in the double mutant. Compared with the nativestructure, no significant conformational differences were observed in thestructures of the mutant proteins. We conclude that the structural anddynamic integrity of the region around these mutations is of crucialimportance to the photocycle and suggest that the hinge-bending propertiesof Gly(51) may also play a role in PAS domain proteins where it is one ofthe few conserved residues.

• Bibikov SI, Barnes LA, Gitin Y, Parkinson JS
• Domain organization and flavin adenine dinucleotide-binding determinantsin the aerotaxis signal transducer Aer of Escherichia coli.
• Proc Natl Acad Sci U S A. 2000; 97: 5830-5
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• Aerotactic responses in Escherichia coli are mediated by the membranetransducer Aer, a recently identified member of the superfamily of PASdomain proteins, which includes sensors of light, oxygen, and redox state.Initial studies of Aer suggested that it might use a flavin adeninedinucleotide (FAD) prosthetic group to monitor cellular redox changes. Totest this idea, we purified lauryl maltoside-solubilized Aer protein byHis-tag affinity chromatography and showed by high performance liquidchromatography, mass spectrometry, and absorbance spectroscopy that itbound FAD noncovalently. Polypeptide fragments spanning the N-terminal 290residues of Aer, which contains the PAS motif, were able to bind FAD.Fusion of this portion of Aer to the flagellar signaling domain of Tsr,the serine chemoreceptor, yielded a functional aerotaxis transducer,demonstrating that the FAD-binding portion of Aer is sufficient foraerosensing. Aerotaxis-defective missense mutants identified two regions,in addition to the PAS domain, that play roles in FAD binding. Thoseregions flank a central hydrophobic segment needed to anchor Aer to thecytoplasmic membrane. They might contact the FAD ligand directly orstabilize the FAD-binding pocket. However, their lack of sequenceconservation in Aer homologs of other bacteria suggests that they playless direct roles in FAD binding. One or both regions probably also playimportant roles in transmitting stimulus-induced conformational changes tothe C-terminal flagellar signaling domain to trigger aerotactic behavioralresponses.

• van den Berg WA, Hagen WR, van Dongen WM
• The hybrid-cluster protein ('prismane protein') from Escherichia coli.Characterization of the hybrid-cluster protein, redox properties of the[2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADHoxidoreductase containing FAD and [2Fe-2S].
• Eur J Biochem. 2000; 267: 666-76
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• Hybrid-cluster proteins ('prismane proteins') have previously beenisolated and characterized from strictly anaerobic sulfate-reducingbacteria. These proteins contain two types of Fe/S clusters unique inbiological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster,which can attain four redox states. Genomic sequencing reveals that genesencoding putative hybrid-cluster proteins are present in a range ofbacterial and archaeal species. In this paper we describe the isolationand spectroscopic characterization of the hybrid-cluster protein fromEscherichia coli. EPR spectroscopy shows the presence of a hybrid clusterin the E. coli protein with characteristics similar to those in theproteins of anaerobic sulfate reducers. EPR spectra of the reduced E. colihybrid-cluster protein, however, give evidence for the presence of a[2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding thehybrid-cluster protein in E. coli and other facultative anaerobes occurs,in contrast with hcp genes in obligate anaerobic bacteria and archaea, ina small operon with a gene encoding a putative NADH oxidoreductase. ThisNADH oxidoreductase was also isolated and shown to contain FAD and a[2Fe-2S] cluster as cofactors. It catalysed the reduction of thehybrid-cluster protein with NADH as an electron donor. Midpoint potentials(25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicatethat electrons derived from the oxidation of NADH (Em NADH/NAD+ couple:-320 mV) are transferred along the [2Fe-2S] cluster of the NADHoxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of thehybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85and +365 mV for the three redox transitions). The physiological functionof the hybrid-cluster protein has not yet been elucidated. The protein isonly detected in the facultative anaerobes E. coli and Morganella morganiiafter cultivation under anaerobic conditions in the presence of nitrate ornitrite, suggesting a role in nitrate-and/or nitrite respiration.

• Koonin EV, Mushegian AR, Galperin MY, Walker DR
• Comparison of archaeal and bacterial genomes: computer analysis of proteinsequences predicts novel functions and suggests a chimeric origin for thearchaea.
• Mol Microbiol. 1997; 25: 619-37
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• Protein sequences encoded in three complete bacterial genomes, those ofHaemophilus influenzae, Mycoplasma genitalium and Synechocystis sp., andthe first available archaeal genome sequence, that of Methanococcusjannaschii, were analysed using the BLAST2 algorithm and methods for aminoacid motif detection. Between 75% and 90% of the predicted proteinsencoded in each of the bacterial genomes and 73% of the M. jannaschiiproteins showed significant sequence similarity to proteins from otherspecies. The fraction of bacterial and archaeal proteins containingregions conserved over long phylogenetic distances is nearly the same andclose to 70%. Functions of 70-85% of the bacterial proteins and about 70%of the archaeal proteins were predicted with varying precision. Thiscontrasts with the previous report that more than half of the archaealproteins have no homologues and shows that, with more sensitive methodsand detailed analysis of conserved motifs, archaeal genomes become asamenable to meaningful interpretation by computer as bacterial genomes.The analysis of conserved motifs resulted in the prediction of a number ofpreviously undetected functions of bacterial and archaeal proteins and inthe identification of novel protein families. In spite of the generallyhigh conservation of protein sequences, orthologues of 25% or less of theM. jannaschii genes were detected in each individual completely sequencedgenome, supporting the uniqueness of archaea as a distinct domain of life.About 53% of the M. jannaschii proteins belong to families of paralogues,a fraction similar to that in bacteria with larger genomes, such asSynechocystis sp. and Escherichia coli, but higher than that in H.influenzae, which has approximately the same number of genes as M.jannaschii. Certain groups of proteins, e.g. molecular chaperones and DNArepair enzymes, thought to be ubiquitous and represented in the minimalgene set derived by bacterial genome comparison, are missing in M.jannaschii, indicating massive non-orthologous displacement of genesresponsible for essential functions. An unexpectedly large fraction of theM. jannaschii gene products, 44%, shows significantly higher similarity tobacterial than to eukaryotic proteins, compared with 13% that haveeukaryotic proteins as their closest homologues (the rest of the proteinsshow approximately the same level of similarity to bacterial andeukaryotic homologues or have no homologues). Proteins involved intranslation, transcription, replication and protein secretion are mostclosely related to eukaryotic proteins, whereas metabolic enzymes,metabolite uptake systems, enzymes for cell wall biosynthesis and manyuncharacterized proteins appear to be 'bacterial'. A similar prevalence ofproteins of apparent bacterial origin was observed among the currentlyavailable sequences from the distantly related archaeal genus, Sulfolobus.It is likely that the evolution of archaea included at least one majormerger between ancestral cells from the bacterial lineage and the lineageleading to the eukaryotic nucleocytoplasm.

• Stock AM
• Energy sensors for aerotaxis in Escherichia coli: something old, somethingnew.
• Proc Natl Acad Sci U S A. 1997; 94: 10487-9

• Kim M, Mathies RA, Hoff WD, Hellingwerf KJ
• Resonance Raman evidence that the thioester-linked 4-hydroxycinnamylchromophore of photoactive yellow protein is deprotonated.
• Biochemistry. 1995; 34: 12669-72
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• Resonance Raman spectra of the ground state of photoactive yellow protein(PYP), a photoactive pigment found in Ectothiorhodospira halophila, havebeen obtained with excitation at 413.1 nm using a microspinning samplecell. The resonance Raman spectra of the thioester-linked4-hydroxycinnamyl chromophore in the protein are compared with thepreresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and4-hydroxycinnamic acid model compounds at various pH values. Bands at1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionicform of the 4-hydroxycinnamyl phenyl thioester are shown to becharacteristic for the deprotonation of the chromophore. The observationof bands in PYP exhibiting very similar frequency and intensity patternsprovides strong evidence that the chromophore in PYP is stabilized as aphenolate anion at pH 7.4, in support of conclusions from crystallographicstudies. Furthermore, the insensitivity of the PYP Raman spectrum toplacement of the protein in D2O buffer is consistent with the absence ofthe exchangeable phenolic proton on the cinnamyl chromophore. Theseresults establish the feasibility of elucidating the molecular mechanismof light-to-information transduction by this new photosensory pigment withresonance Raman spectroscopy.