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miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen. Graphical abstract

International audience; ECFP, the first usable cyan fluorescent protein (CFP), was obtained by adapting the tyrosine-based chromophore environment in green fluorescent protein to that of a tryptophan-based one. This first-generation CFP was superseded by the popular Cerulean, CyPet, and SCFP3A that were engineered by rational and random mutagenesis, yet the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were serendipitously corrected in the third-generation CFP mTurquoise and its successors without an obvious rationale. We show here that the evolution process had unexpectedly remodeled the chromophore environment in second-generation CFPs so they would accommodate a different isomer, whose formation is favored by acidic pH or light irradiation and which emits fluorescence much less efficiently. Our results illustrate how fluorescent protein engineering based solely on fluorescence efficiency optimization may affect other photophysical or physicochemical parameters and provide novel insights into the rational evolution of fluorescent proteins with a tryptophan-based chromophore.

Membrane proteins constitute about one third of proteins encoded by all genomes, but only a small percentage have their structures deposited in the Protein Data Bank. One bottleneck in the pipeline from expression to structure determination is the identification of detergents that maintain the protein in a soluble, stable, and active state. Here, we describe a small-scale automated procedure to easily and rapidly screen detergents for the solubilization and purification of membrane proteins, to perform detergent exchange, or to identify conditions preserving protein interactions in complexes. Hundreds of conditions can be tested in a few hours to select detergents that keep proteins folded and nonaggregated, from single membrane preparations of cells overexpressing the protein(s) of interest. Thirty-one prokaryotic, eukaryotic, and viral membrane proteins were analyzed by our small-scale procedure to identify the best-associated detergents. Examples of results obtained with a bitopic and multitopic membrane proteins and membrane protein complexes are presented in more detail. DDM, DM, DMNG, TritonX-100, LAPAO, and Fos-12 appeared effective for successful membrane solubilization and protein purification of most selected targets. Eukaryotic proteins are in general more difficult to extract and purify from Escherichia coli membranes than prokaryotic proteins. The protocol has been developed for His-tagged proteins, but can readily be adapted to other affinity tags by adjusting the chromatography resin and the buffer composition.

Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (

Cryoprotection of a protein crystal by addition of small-molecule compounds may sometimes affect the structure of its active site. The spectroscopic and structural effects of the two cryoprotectants glycerol and ethylene glycol on the cyan fluorescent protein Cerulean were investigated. While glycerol had almost no noticeable effect, ethylene glycol was shown to induce a systematic red shift of the UV–vis absorption and fluorescence emission spectra. Additionally, ethylene glycol molecules were shown to enter the core of the protein, with one of them binding in close vicinity to the chromophore, which provides a sound explanation for the observed spectroscopic changes. These results highlight the need to systematically record spectroscopic data on crystals of light-absorbing proteins and reinforce the notion that fluorescent proteins must not been seen as rigid structures.

The green fluorescent protein (GFP) from the jellyfish Aequoria victoria has been shown to dimerize at high concentrations, which could lead to artefacts in imaging experiments. To ensure a truly monomeric state, an A206K mutation has been introduced into most of its widely used variants, with minimal effect on the spectroscopic properties. Here, the first structure of one of these variants, the cyan fluorescent protein mTurquoise, is presented and compared with that of its dimeric version mTurquoise-K206A. No significant structural change is detected in the chromophore cavity, reinforcing the notion that this mutation is spectroscopically silent and validating that the structural analysis performed on dimeric mutants also applies to monomeric versions. Finally, it is explained why cyan versions of GFP containing the Y66W and N146I mutations do not require the A206K mutation to prevent dimerization at high concentrations.

To make progress in understanding peptidoglycan metabolism, we will reconstitute in vitro the assembly process and the molecular machineries that carry out this formidable task. We review here the reports of isolation of complexes comprising penicillin-binding proteins (PBPs), the enzymes that synthesize the peptidoglycan from its lipid-linked precursor.

Obtaining sequence-specific assignments remains a major bottleneck in solution NMR investigations of supramolecular structure, dynamics and interactions. Here we demonstrate that resonance assignment of methyl probes in high molecular weight protein assemblies can be efficiently achieved by combining fast NMR experiments, residue-type-specific isotope-labeling and automated site-directed mutagenesis. The utility of this general and straightforward strategy is demonstrated through the characterization of intermolecular interactions involving a 468-kDa multimeric aminopeptidase, PhTET2.

Enhanced Green Fluorescent Protein (EGFP) is a variant of wild-type Green Fluorescent Protein from the jellyfish Aequorea victoria, whose mutations S65T and F64L increase brightness and folding efficiency. EGFP is extensively used in cell biology and biochemistry as a colocalization or expression reporter. Surprisingly, the structure of this very popular protein has not been determined yet. We report here its crystallographic structure at 1.5Å resolution which shows significant differences in the vicinity of residue 64 and of the chromophore. In particular, two conformations are observed for the key residue glutamic acid 222, in apparent contradiction with the single fluorescence lifetime of the protein. We then show that X-ray induced decarboxylation of Glu222 during diffraction data collection results in the disruption of a hydrogen-bond network near the chromophore. Using single-crystal microspectrophotometry, we demonstrate that this correlates with a significant loss of the fluorescence properties. We thus propose a mechanism of bleaching of the protein at low temperature. Taken together, these two sets of results highlight the stabilizing role of Glu222 to the chromophore cavity of EGFP.

Enhanced cyan fluorescent protein (ECFP) and its variant Cerulean are genetically encoded fluorophores widely used as donors in FRET-based cell imaging experiments. First, we have confirmed through denaturation experiments that the double-peak spectroscopic signature of these fluorescent proteins originates from the indole ring of the chromophore. Then, to explain the improvement in the fluorescence properties of Cerulean compared to those of ECFP, we have determined the high-resolution crystal structures of these two proteins at physiological pH and performed molecular dynamics simulations. In both proteins, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore and, as our simulations suggest, they interconvert on a nanosecond time scale. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching, albeit only intermittently. As a consequence, the probability of nonradiative decay is significantly decreased. Our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins and provide insights that should allow the design of mutants with enhanced optical properties.

Synchrotrons are now producing thousands of macromolecular structures each year. The need for complementary techniques available on site has progressively emerged, either to assess the relevance of the structure of a protein or to monitor changes that may occur during X-ray diffraction data collection. Microspectrophotometers in the UV-visible absorbance or fluorescence mode have evolved over the past few decades to become the instruments of choice to perform such tests. Described here are recent improvements to the microspectrophotometer of the so-called Cryobench laboratory located at the European Synchrotron Radiation Facility, Grenoble, France. Optical and mechanical properties have been enhanced so as to record better spectra on smaller samples. A device has been implemented to measure the signal decay of fluorescent samples, either in the crystalline or in the solution state. Recording of the fluorescence lifetime in addition to the steady-state fluorescence emission spectrum allows precise monitoring of the fluorescent sample under study. The device consists of an adaptation of a commercially available time-correlated single-photon-counting (TCSPC) system. A method to record and analyze series of TCSPC histograms, e. g. collected as a function of temperature, is described. To validate the instruments, fluorescence lifetimes of fluorescent small molecules or proteins in the crystalline or solution state, at room and cryo temperatures, have been measured. Lifetimes of a number of fluorescent proteins of the GFP family were generally found to be shorter in crystals than in solution, and slightly longer at cryo temperatures than at ambient temperature. The possibility of performing fluorescence lifetime measurements on crystals at synchrotron facilities widens the variety of spectroscopic techniques complementing X-ray diffraction on macromolecular crystallography beamlines. ispartof: Journal of Applied Crystallography vol:40 pages:1105-1112 status: published

International audience; Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality.

DivIB, DivIC and FtsL are bacterial proteins essential for cell division, which show interdependencies for their stabilities and localization. We have reconstituted in vitro a trimeric complex consisting of the recombinant extracellular domains of the three proteins from Streptococcus pneumoniae. The extracellular domain of DivIB was found to associate with a heterodimer of those of DivIC and FtsL. The heterodimerization of DivIC and FtsL was artificially constrained by fusion with interacting coiled-coils. Immunofluorescence experiments showed that DivIC is always localized at mid-cell, in contrast to DivIB and FtsL, which are co-localized with DivIC only during septation. Taken together, our data suggest that assembly of the trimeric complex DivIB/DivIC/FtsL is regulated during the cell cycle through controlled formation of the DivIC/FtsL heterodimer.

Summary Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a d,d-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.

sigma(S) (RpoS), the master regulator of the general stress response in Escherichia coli, is a model system for regulated proteolysis in bacteria. sigma(S) turnover requires ClpXP and the response regulator RssB, whose phosphorylated form exhibits high affinity for sigma(S). Here, we demonstrate that recognition by the RssB/ClpXP system involves two distinct regions in sigma(S). Region 2.5 of sigma(S) (a long alpha-helix) is sufficient for binding of phosphorylated RssB. However, this interaction alone is not sufficient to trigger proteolysis. A second region located in the N-terminal part of sigma(S), which is exposed only upon RssB-sigma(S) interaction, serves as a binding site for the ClpX chaperone. Binding of the ClpX hexameric ring to sigma(S)-derived reporter proteins carrying the ClpX-binding site (but not the RssB-binding site) is also not sufficient to commit the protein to degradation. Our data indicate that RssB plays a second role in the initiation of sigma(S) proteolysis that goes beyond targeting of sigma(S) to ClpX, and suggest a model for the sequence of events in the initiation of sigma(S) proteolysis.

International audience; Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.

Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.

There is increasing interest in applying NMR spectroscopy to the study of large protein assemblies. Development of methyl-specific labeling protocols combined with improved NMR spectroscopy enable nowadays studies of proteins complexes up to 1 MDa. For such large complexes, the major interest lies in obtaining structural, dynamic and interaction information in solution, which requires sequence-specific resonance assignment of NMR signals. While such analysis is quite standard for small proteins, it remains one of the major bottlenecks when the size of the protein increases. Here, we describe implementation and latest improvements of SeSAM, a fast and user-friendly approach for assignment of methyl resonances in large proteins using mutagenesis. We have improved culture medium to boost the production of methyl-specifically labeled proteins, allowing us to perform small-scale parallel production and purification of a library of (13)CH3-specifically labeled mutants. This optimized protocol is illustrated by assignment of Alanine, Isoleucine, and Valine methyl groups of the homododecameric aminopeptidase PhTET2. We estimated that this improved method allows assignment of ca. 100 methyl cross-peaks in 2 weeks, including 4 days of NMR time and less than 2 k€ of isotopic materials.

International audience; Tetrahedral (TET) aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model. Point mutations allowed the capture of a stable, catalytically active precursor. Small angle x-ray scattering revealed that it is a dimer whose architecture in solution is identical to that determined by x-ray crystallography within the fully assembled TET particle. Small angle x-ray scattering also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild-type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis, and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates. Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo.

International audience; The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

International audience; In this report we describe a rapid, simple, and efficient method for large scale purification of linear plasmid DNA to answer demand from high throughput gene cloning. The process is based on the separation of the linear vector from small DNA fragments by anion exchange chromatography. Gene cloning experiments by restriction/ligation or the In-Fusion™ technique confirmed the high quality of the linearized vector as 100% of the genes were successfully cloned.

Background Streptococcus pneumoniae is a widely distributed commensal Gram-positive bacteria of the upper respiratory tract. Pneumococcal colonization can progress to invasive disease, and thus become lethal, reason why antibiotics and vaccines are designed to limit the dramatic effects of the bacteria in such cases. As a consequence, pneumococcus has developed efficient antibiotic resistance, and the use of vaccines covering a limited number of serotypes such as Pneumovax® and Prevnar® results in the expansion of non-covered serotypes. Pneumococcal surface proteins represent challenging candidates for the development of new therapeutic targets against the bacteria. Despite the number of described virulence factors, we believe that the majority of them remain to be characterized. This is the reason why pneumococcus invasion processes are still largely unknown. Results Availability of genome sequences facilitated the identification of pneumococcal surface proteins bearing characteristic motifs such as choline-binding proteins (Cbp) and peptidoglycan binding (LPXTG) proteins. We designed a medium throughput approach to systematically test for interactions between these pneumococcal surface proteins and host proteins (extracellular matrix proteins, circulating proteins or immunity related proteins). We cloned, expressed and purified 28 pneumococcal surface proteins. Interactions were tested in a solid phase assay, which led to the identification of 23 protein-protein interactions among which 20 are new. Conclusions We conclude that whether peptidoglycan binding proteins do not appear to be major adhesins, most of the choline-binding proteins interact with host proteins (elastin and C reactive proteins are the major Cbp partners). These newly identified interactions open the way to a better understanding of host-pneumococcal interactions.

International audience; Sometimes less is more: [13C1H3]methyl isotopomers can be biosynthetically incorporated specifically into the pro-S methyl groups of leucine and valine residues in large protein assemblies within a perdeuterated background by using an acetolactate precursor. This stereospecific labeling strategy considerably enhances NMR spectra for large protein assemblies.

A major challenge in structural biology remains the identification of protein constructs amenable to structural characterization. Here, we present a simple method for parallel expression, labeling, and purification of protein constructs (up to 80 kDa) combined with rapid evaluation by NMR spectroscopy. Our approach, which is equally applicable for manual or automated implementation, offers an efficient way to identify and optimize protein constructs for NMR or X-ray crystallographic investigations.

Transposition of insertion sequences (IS) is an enzyme-mediated process that only occurs in a minority of cells within a bacterial culture. Transposition is thus a rare event, but transposition frequency may vary depending on experimental conditions. For instance in a rich broth, IS elements are known to transpose during stationary phase but not during exponential growth. Using a reporter system which involves the activation of the cryptic bgl operon in Escherichia coli, we show that the frequency of IS1 transposition is a function of glucose concentration in the growth medium, it is increased by streptomycin amounts that are below minimum inhibitory concentration (sub-MIC) and is inhibited in an rpsL150 strain with high translation accuracy. Since starved cells are known to enhance ribosome frameshifting, our data suggests that growth conditions applied in this study could affect IS1 transposition by increasing translation infidelity.

FtsW and RodA are homologous integral membrane proteins involved in bacterial cell division and cell growth, respectively. Both proteins from Streptococcus pneumoniae were overexpressed in Escherichia coli as N-terminal His-tagged fusions. Their membrane addressing in E. coli was demonstrated by cell fractionation and was confirmed for FtsW by immunolocalization. Recombinant FtsW and RodA were solubilized from membranes using 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO). The detergent was exchanged to polyoxyethylene 8 lauryl ether (C12E8) during one-step purification procedure by Co(2+)-affinity chromatography. This procedure yielded 50-150 microg protein per litre of culture. Both proteins are likely to be folded as they are resistant to trypsin digestion and could be incorporated into reconstituted lipid vesicles.

Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh β-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein., Cyan variants of green fluorescent protein (CFPs) are widely used as donors in FRET experiments. Here, a new CFP, mTurquoise2, is developed, which displays a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime.