Institut de Biologie StructuraleGrenoble / France


New issue for the IBS Newsletter

Find the January 2018 issue (in french only).

Capture of a «phantom » state of green fluorescent proteins

Green fluorescent proteins (GFPs) are genetically encoded markers allowing the localization of down to individual proteins by optical microscopy. Their fluorophore is formed from three amino acids and is located at the center of a beta barrel that protects it from the environment. GFPs have high absorption coefficient and fluorescence yield, but only moderate photostability. They can reversibly form temporary nonfluorescent “dark” states before definitely losing fluorescence after about 100000 excitation/emission cycles.
At the IBS, the Pixel team has previously solved the structures of some of such dark states. All of them showed considerable chemical modifications which raised the question as to their irreversibility. Therefore, it was suspected that a reversible gateway state should exist the knowledge of which would allow to improve the GFP’s behavior. But little was known on this putative « phantom » state: its chemical character (triplet, radical, biradical ?), its yield (percents, permills ?), its lifetime (nano-, micro-, millisecondes ?), its reactivity (oxidative, reductive, both ?), its absorption spectrum (UV, VIS, IR ?).
Thanks to a collaboration with the team of Klaus BRETTEL at I2BC (CEA Saclay), the Pixel team of the DYNAMOP group at the IBS exposed the paradigmatic fluorescent protein EGFP to a study by transient absorption spectroscopy. This technique has previously been applied to fluorescent proteins very little. Pushing it to its resolution limits allowed the detection of the spectral signature of the « phantom » dark state and its characterization by answering all of the above questions. These findings open the way for a better understanding of the photophysical functioning of fluorescent proteins and fuel the hope for finding keys to their further optimization.

A Long-Lived Triplet State Is the Entrance Gateway to Oxidative Photochemistry in Green Fluorescent Proteins. Byrdin M, Duan C, Bourgeois D, Brettel K. Journal of the American Chemical Society;. doi: 10.1021/jacs.7b12755

How bacteria converse in floating biofilms

Biofilms are bacterial communities with high antibiotic resistance. Within biofilms, bacteria exchange information chemically - a mechanism called quorum-sensing. Researchers at the Institute de Biologie Structurale in Grenoble, the University of the Mediterranean in Marseille and the Jacobs University in Bremen have shown that the gram-negative pathogen Providencia stuartii forms floating communities within which adjacent cells are in apparent contact,before depositing as canonical surface-attached biofilms. Because porins are the most abundant proteins in the outer membrane of gram-negative bacteria, they hypothesized that they could be involved in cell-to-cell contact and undertook a structure-function relationship study on the two porins of P. stuartii, Omp-Pst1 and Omp-Pst2. The crystal structures reveal that these porins can selfassociate through their extracellular loops, forming dimers of trimers (DOTs) that could enable cell-to-cell contact within floating communities. Support for this hypothesis was obtained by studying the porin-dependent aggregation of liposomes and model cells. The observation that facing channels are open in the two porin structures suggests that DOTs could not only promote cell-to-cell contact but also contribute to intercellular communication. Proteins involved in this interaction could be new targets in the fight against biofilms.

Porin self-association enables cell-to-cell contact in Providencia stuartii floating communities. El-Khatib M, Nasrallah C, Lopes J, Tran QT, Tetreau G, Basbous H, Fenel D, Gallet B, Lethier M, Bolla JM, Pagès JM, Vivaudou M, Weik M, Winterhalter M, Colletier JP. Proceedings of the National Academy of Sciences of the United States of America. 2018 Feb 23. pii: 201714582. doi: 10.1073/pnas.1714582115.

Bacterial pathogens can reprogram target cells by influencing epigenetic factors

The type III secretion system (T3SS) is a complex nanomachine used by numerous Gram-negative bacteria to inject toxins directly into target cells. Its architecture resembles a syringe, and toxins are believe to travel through its interior. One key aspect of the T3SS is the translocon, a complex of two membrane proteins that are synthesized within the bacterial cytoplasm, transported through the interior of the needle, and subsequently inserted directly into the membrane of the eukaryotic cell, allowing toxin passage. In this work IBS scientists and their collaborators from BIG and London Imperial College showed that insertion of the translocon proteins (PopB and PopD) by the human pathogen Pseudomonas aeruginosa into target membranes engenders epigenetic modifications on histone H3 as a consequence of ion exchange through the formed pore. This thus indicates, for the first time, that the translocon acts not only as a pore, but also as a bona fide virulence factor.

Pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon alters the host epigenome. Laurent Dortet, Charlotte Lombardi, François Cretin, Andréa Dessen, Alain Filloux. Nature Microbiology 2018 Feb 5. doi: 10.1038/s41564-018-0109-7

Analytical Description of NMR Relaxation Highlights Correlated Dynamics in Intrinsically Disordered Proteins

The dynamic fluctuations of intrinsically disordered proteins (IDPs) define their function. Although experimental nuclear magnetic resonance (NMR) relaxation reveals the motional complexity of these highly flexible proteins, the absence of physical models describing IDP dynamics hinders their mechanistic interpretation. Combining molecular dynamics simulation and NMR, the researchers of the FDP group (Protein Dynaics and Flexibility by NMR) introduce a framework in which distinct motions are attributed to local libration, backbone dihedral angle dynamics and longer-range tumbling of one or more peptide planes. This model provides unique insight into segmental organization of dynamics in IDPs and allows them to investigate the presence and extent of the correlated motions that are essential for function.

Analytical Description of NMR Relaxation Highlights Correlated Dynamics in Intrinsically Disordered Proteins. Salvi N, Abyzov A, Blackledge M. Angewandte Chemie International Edition England ;56(45):14020-14024.

Antibiotics and radical-based chemistry: the 1,2-diol dehydratase AprD4 from the inside

New sources of antibiotics are required to fight against multidrug-resistant pathogens. Natural product biosynthetic pathways are a vast source of inspiration to develop new efficient and environment-friendly chemical synthesis processes. Radical-based chemistry, using high-energy intermediates can afford difficult reactions in water. ‘Radical SAM’ enzymes control such intermediates to perform regio- and stereo-specific reactions. The crystal structure of the radical SAM 1,2-diol dehydratase AprD4, determined by the Metalloproteins unit at IBS in collaboration with the group of Pr. Qi Zhang at Fudan University (Shanghai, China), has revealed that the remarkable tridimensional arrangement at its active site, while keeping substrate-specificity, gives the radical intermediate enough freedom to adopt different conformations, in order to release a specific water molecule. This modification makes certain aminoglycoside antibiotics insensitive to the most common mechanisms of resistance toward this family of antimicrobial agents.

1,2-diol dehydration by the radical SAM enzyme AprD4 - a matter of proton circulation and substrate flexibility. Liu WQ, Amara P, Mouesca JM, Ji X, Renoux O, Martin L, Zhang C, Zhang Q, Nicolet Y. Journal of the American Chemical Society 2018 Jan 4. doi: 10.1021/jacs.7b10501. [Epub ahead of print]