Marjolaine Noirclerc-Savoye

Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.

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.

The d,d‐carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae

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.

Sequential recognition of two distinct sites in σS by the proteolytic targeting factor RssB and ClpX

σS (RpoS), the master regulator of the general stress response in Escherichia coli, is a model system for regulated proteolysis in bacteria. σS turnover requires ClpXP and the response regulator RssB, whose phosphorylated form exhibits high affinity for σS. Here, we demonstrate that recognition by the RssB/ClpXP system involves two distinct regions in σS. Region 2.5 of σS (a long α‐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 σS, which is exposed only upon RssB–σS interaction, serves as a binding site for the ClpX chaperone. Binding of the ClpX hexameric ring to σS‐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 σS proteolysis that goes beyond targeting of σS to ClpX, and suggest a model for the sequence of events in the initiation of σS proteolysis.

An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging

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 heme oxygenase responsible for BV biosynthesys and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared to monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labeling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumors in whole mice. Our work shows promise in the application of IFPs for protein labeling and in vivo imaging.

Stabilizing Role of Glutamic Acid 222 in the Structure of Enhanced Green Fluorescent Protein.

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.

Intrinsic Dynamics in Ecfp and Cerulean Control Fluorescence Quantum Yield.

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.

In vitro reconstitution of a trimeric complex of DivIB, DivIC and FtsL, and their transient co‐localization at the division site in Streptococcus pneumoniae

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.

Advances in spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements

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.

New adhesin functions of surface-exposed pneumococcal proteins

BackgroundStreptococcus 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.ResultsAvailability 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.ConclusionsWe 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.

A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies

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.