IBS - Institut de Biologie Structurale - Grenoble / France

Intrinsically disordered tardigrade proteins self-assemble into fibrous gels in response to environmental stress

PELLEQUER Jean-Luc — 2026-01-27T09:27:28Z

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade-unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS-8 from Hypsibius exemplaris. AFM imaging shows that the protein successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature-dependent manner. NMR reveals that the helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel.
The AFM images was performed by Jean-Marie Teulon from samples prepared by Anas Malki. Enhancement of AFM images using the Laplacian weight filter was performed by Wendy Chen.

Malki A, Teulon J-M, Camacho Zarco A, Chen S-wW, Adamski W, Maurin D, Salvi N, Pellequer J-L and Blackledge M (2022) Intrinsically disordered tardigrade proteins self-assemble into fibrous gels in response to environmental stress. Angew. Chem. Int. Ed. 61 : e202109961.

AFM-ASSEMBLY

PELLEQUER Jean-Luc — 2025-04-03T10:59:12Z

Pellequer J-L (2024) Perspectives toward an integrative structural biology pipeline with atomic force microscopy topographic images. J. Mol. Recognit. 37: e3102.

AFM-Assembly is a suite of scripts and programs for assembling three-dimensional structures (PDB format) under the experimental constraint of the topographic surface of a molecule of interest.

AFM-Assembly bridges the gap between high-resolution AFM imaging of single, isolated particles and three-dimensional molecule assembly at the atomic level.

Admittedly, the resolution of an AFM topographic surface is not sufficient to directly constrain the positioning of atoms/residues from a topographic surface. However, it is entirely possible to assemble different pieces of a structure, or complex, from PDB files, all under the experimental constraint of single molecules.

The big difference between AFM imaging and electron microscopy is the unique signal-to-noise ratio of an AFM topography, which allows us to “see” the global conformation of a single molecule (with a practical resolution of nm).

AFM-Assembly was originally developed by Minh-hieu Trinh and extended in its current form by Rui M. Chaves for fitting and assembly.

Root growth arrest, rigidity and metal stress

PELLEQUER Jean-Luc — 2023-11-01T10:27:18Z

We investigated the change in stiffness of the external primary cell wall of living Arabidopsis thaliana seedlings in the presence of metallic stress using atomic force microscopy. Results reveal for the first time the uncoupling between mechanical response (CW stiffening) and root extension arrest. Molecular synergy has also been demonstrated in the physiological response to stress, where the Aluminum stress response enhances the Iron stress response via the organic acid molecule: malate.

Kaur H, Teulon J-M, Godon C, Desnos T, Chen S-wW and Pellequer J-L (2024) Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al. Plant Cell Environ. 47:574–584.

Native cristals from Bacillus thuringiensis

PELLEQUER Jean-Luc — 2023-09-11T11:31:30Z

Cry11Aa and Cyt1Aa are two pesticidal toxins produced by Bacillus thuringiensis subsp. israelensis. To better understand the nature of their oligomers in toxic actions and synergistic effects, we used atomic force microscopy to probe the surfaces of their native crystals, and employed the L-weight filter to enhance structural features.

Images of these crystals can be found in several publications in collaboration with Jacques-Philippe Colletier's SNAX team at IBS. AFM images were obtained by Jean-Marie Teulon and image processing was carried out by Shu-wen W. Chen.

Tetreau G, Banneville A-S, Andreeva EA, Brewster AS, Hunter MS, Sierra RG, Teulon J-M, Young ID, Burke N, Gruenewald T, Beaudouin J, Snigireva I, Fernandez-Luna MT, Burt A, Park H-W, Signor L, Bafna JA, Sadir R, Fenel D, Boeri-Erba E, Bacia M, Zala N, Laporte F, Després L, Weik M, Boutet S, Rosenthal M, Coquelle N, Burghammer M, Cascio D, Sawaya MR, Winterhalter M, Gratton E, Gutsche I, Federici B, Pellequer J-L, Sauter NK and Colletier J-P (2020) Serial femtosecond crystallography drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade, from in vivo crystallization to cell lysis. Nat. Comm. 11: 1153.

Tetreau G, Sawaya MR, De Zitter E, Andreeva EA, Banneville A-S, Schibrosky N, Coquelle N, Brewster AS, Schiro G, Grünbein M-L, Kovacs GN, Hunter MS, Kloos M, Sierra RG, Qiao P, Bideshi D, Young ID, Zala N, Engilberge S, Gorel A, Signor L, Teulon J-M, Bielicki J, Bean R, Letrun R, Batyuk A, Snigireva I, Fenel D, Schubert R, Laporte F, Després L, Bacia M, Girard E, Roux-Gossart A, Chapelle C, Maury O, Ling WL, Boutet S, Mancuso A, Barends TRM, Pellequer JL, Park H-W, Laganowsky AD, Rodriguez J, Burghammer M, Shoeman RL, Doak RB, Weik M, Sauter NK, Federici B, Cascio D, Schlichting I and Colletier J-P (2022) De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures by serial femtosecond crystallography on nanocrystals. Nat. Comm. 13: 4376.

Chen SWW, Teulon JM and Pellequer JL (2023) Cry11Aa and Cyt1Aa exhibit different structural orders in crystal topography. J. Mol. Recogn. 36: e3047.

Optimized protocol for determining plant root stiffness

PELLEQUER Jean-Luc — 2023-09-11T08:26:22Z

Stiffness plays a central role in plant cell extension. We have optimized a protocol for detecting stiffness changes on the outer epidermal cell wall of living plant roots using atomic force microscopy (AFM). A protocol provides general instructions for collecting force-distance curves and analyzing stiffness using a contact-based mechanical model. With this protocol and initial AFM training, a user is able to perform indentation experiments on 4- and 5-day-old Arabidopsis thaliana and determine stiffness properties.

Kaur H, Teulon J-M, Foucher A-E, Fenel D, Chen S-wW, Godon C, Desnos T and Pellequer J-L (2023) Measuring external primary cell wall elasticity of seedling roots using atomic force microscopy. STAR Protoc. 4: 102265.

1995-2001 (TSRI, La Jolla)

PELLEQUER Jean-Luc — 2023-05-05T11:24:25Z

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– Hsiung YG, Chang HC, Pellequer J-L, LaValle R and Wittenberg C (2001) The F-box protein Grr1 interacts with phosphorylated targets via the cationic surface of its Leucine-Rich Repeat. Mol. Cell Biol. 21: 2506-2520. [HAL]
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– Pellequer J-L, Chen S-wW, Feeney AJ, Zhao B, Kao H-I, Karu AE, Li K, Li QX and Roberts VA (2001) Architecture of antibody binding sites for polunuclear aromatic hydrocarbons. Nuclear Site Remediation. First accomplishments of the environmental management science program. Eller PG and Heineman WR, American Chemical Society. 778: 398-416. [HAL]

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– Gale AJ,Pellequer JL, Getzoff ED and Griffin JH (2000) Structural basis for hemophilia A caused by mutations in the C domains of blood coagulation factor VIII. Thromb. Haemost. 83: 78-85. [HAL]
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– Pellequer J-L, Gale AJ and Getzoff ED (2000) Blood coagulation: The outstanding hydrophobic residues. Curr. Biol. 10: R237-R240. [HAL]

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– Pellequer J-L, Gale AJ, Getzoff ED and Griffin JH (2000) Three-dimensional model of the coagulation factor Va bound to activated protein C. Thromb. Haemost. 84: 849-857. [HAL]
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– Pellequer J-L, Zhao B, Kao H-I, Bell CW, Li K, Li QX, Karu AE and Roberts VA (2000) Stabilization of bound polycyclic aromatic hydrocarbons by a pi-cation interaction. J. Mol. Biol. 302: 691-699. [HAL]

– Pellequer J-L, Brudler R and Getzoff ED (1999) Biological sensors: More than one way to sense oxygen. Curr. Biol. 9: R416-R418. [HAL]

– Pellequer J-L, Chen S-wW, Roberts VA, Tainer JA and Getzoff ED (1999) 3.0.CO;2-9/full" class="spip_out" rel="external">Unraveling the effect of changes in conformation and compactness at the antibody VL-VH interface upon antigen binding. J. Mol. Recognit. 12: 267-275. [HAL]

– Pellequer J-L, Gale AJ, Griffin JH and Getzoff ED (1998) Homology models of the C domains of blood coagulation factors V and VIII: A proposed membrane binding mode for FV and FVIII C2 domains. Blood Cells Mol. Dis. 24: 448-461.

– Pellequer J-L, Wager-Smith KA, Kay SA and Getzoff ED (1998) Photoactive yellow protein: A structural prototype for the three-dimensional fold of the PAS domain superfamily. Proc. Natl. Acad. Sci. USA 95: 5884-5890. [HAL]

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– Coombs GS, Bergstrom RC, Pellequer J-L, Baker SI, Navre M, Tainer JA, Madison EL and Corey DR (1998) Substrate specificity of Prostate-specific antigen (PSA). Chem. Biol. 5: 475-488. [HAL]
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– Pellequer J-L and Chen S-wW (1997) Does conformational free energy distinguish loop conformations in proteins? Biophys. J. 73: 2359-2375. [HAL]

The trimechanic theory

PELLEQUER Jean-Luc — 2023-01-10T15:27:25Z

A new version of the contact-based mechanical model with the strict application of Sneddon's principles.

The trimechanic theory is the very concept of composite nanomechanics underlying the restoring mechanism
of the material under an external compression. It provides a disentanglement of the linear and tip-shape related mechanical responses at various indentation depths.

The trimechanic theory applies to all contact-based mechanical models with a power-law force–depth relationship. The perspective of this research is that stiffness measurement will not remain at a level of global assessment, but will go further to link elastic behaviors with the substructure of the nanomaterial.

– Chen SWW, Teulon JM, Kaur H, Godon C and Pellequer JL (2023) Nano-structural stiffness measure for soft biomaterials of heterogeneous elasticity. Nanoscale Horiz. 8: 75-82. [HAL]

Atomic force microscopes (AFM)

PELLEQUER Jean-Luc — 2022-12-05T12:29:30Z

Multimode 8, Nanoscope V (Bruker)

Dimension 3100, Nanoscope V (Bruker)

Additional instruments

PELLEQUER Jean-Luc — 2022-12-05T12:26:33Z

Epifluorescence Microscope TE2000 (Nikon)

UV-ozone lamp, home made!

Peltier-cooled incubateur Memmert IPP100+ with a full-spectrum LED light 45 W

Compact coating Unit 010, magnetron sputter head for fine-grained sputtering
The interaction between a magnetic field and an electric field causes the electrons to spiral in the vicinity of the target surface (usually metal: Al, Au, C, Cr, Cu, Ni, Ti) thereby increasing the probability that electrons will strike the argon gas to generate ions. The generated ions collide with the target surface under the action of an electric field to sputter the target coated surface (usually mica).

SuperDry cabinet to keep your sample dry. Target RH by default at 5%

T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria

PELLEQUER Jean-Luc — 2022-12-05T11:59:05Z

We combined in vivo, in vitro, and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix.
AFM imaging was performed by Jean-Marie Teulon and TEM images were obtained by Daphna Fenel from samples provided by Viviana Job.

Lombardi C, Tolchard J, Bouillot S, Signor L, Gebus C, Liebl D, Fenel D, Teulon J-M, Brock J, Habenstein B, Pellequer J-L, Faudry E, Loquet A, Attrée I, Dessen A and Job V (2019) Structural and functional characterization of the Type Three Secretion System (T3SS) needle of Pseudomonas aeruginosa. Front. Microbiol. 10: 573.