Determination of protein structures by two dimensional vibrational spectroscopy isotope dilution experiments

Prior to this work two-dimensional vibrational echo spectroscopy (2D-IR) had been limited to the determination of small peptide structures. This thesis proposes and demonstrates a methodology that allows the extension of the spectroscopic principles gained with small molecules to the determination of structures of more complex systems, in particular, transmembrane helix dimers. Micelle suspensions of pairs of transmembrane domains are prepared with varying levels of heavy (13C=18O) isotope substitutions in the helix peptide backbone at selected locations. Vibrational coupling causes tertiary delocalization of the vibrational excitation and a splitting of the isotopic pair spectral transitions. The ultrafast time resolution of 2D-IR allows one to determine the vibrational frequency correlation of the selected modes and establish a dynamical model of spectra that is adequate to extract vibrational coupling constants from the comparison of spectra at different isotopic concentrations. The frequency correlation function is also capable of indicating the presence of mobile water associated with the labeled residues. The constraints derived from vibrational coupling of the precisely spaced heavy residues leads to determination of an optimized structure from a range of model candidates. The methods developed herein are applicable and transferable to other protein samples. This approach, as described, will identify conformational exchange or any other dynamical phenomena in addition to characterizing the structure of the exchanging species in picoseconds (ps) or longer timescales

Determination of protein structures by two dimensional vibrational spectroscopy isotope dilution experiments

Prior to this work two-dimensional vibrational echo spectroscopy (2D-IR) had been limited to the determination of small peptide structures. This thesis proposes and demonstrates a methodology that allows the extension of the spectroscopic principles gained with small molecules to the determination of structures of more complex systems, in particular, transmembrane helix dimers. Micelle suspensions of pairs of transmembrane domains are prepared with varying levels of heavy (13C=18O) isotope substitutions in the helix peptide backbone at selected locations. Vibrational coupling causes tertiary delocalization of the vibrational excitation and a splitting of the isotopic pair spectral transitions. The ultrafast time resolution of 2D-IR allows one to determine the vibrational frequency correlation of the selected modes and establish a dynamical model of spectra that is adequate to extract vibrational coupling constants from the comparison of spectra at different isotopic concentrations. The frequency correlation function is also capable of indicating the presence of mobile water associated with the labeled residues. The constraints derived from vibrational coupling of the precisely spaced heavy residues leads to determination of an optimized structure from a range of model candidates. The methods developed herein are applicable and transferable to other protein samples. This approach, as described, will identify conformational exchange or any other dynamical phenomena in addition to characterizing the structure of the exchanging species in picoseconds (ps) or longer timescales

Luminescence Quenching of Eu(III) Carboxylates by Cu(II) in a Composite Polymer Xerogel Film

Three Eu(III) luminescent compounds were separately entrapped in a xerogel porous silica matrix and finely ground particles of it were deposited on a glass support with polyvinylacetate (PVAc) as a binder to build a thin film sensor. These 3 devices were immersed in aqueous solutions of Cu(II) and the content of this metal was evaluated by emissionquenching experiments. The sensor containing the highly luminescent antenna chelate of diethylenetriaminepentaacetic acid (dtpa) sensitized with Coumarin120 rendered the largest Stern-Volmer constant (KSV 5 1.49 3 104 M21 ), showing no leaching of the Eu(III) complex to the aqueous solution and a reproducible value of the luminescence ratio between water and Cu(II) solution. The in situ sensor we developed can measure the concentration of Cu(II) in aqueous media down to the ppm level by emission-quenching experiments. This methodology permits a simple calibration of the sensor and an easy to use reusable device.Fil: Barja, Beatriz Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Remorino, Amanda. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentin

Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics

AbstractRecently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability

Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion

International audienceThe spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion

Gradients of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling

International audienceRac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50–100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms

RalB directly triggers invasion downstream Ras by mobilizing the Wave complex

International audienceThe two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies