Dataset Information

Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides.

ABSTRACT: Activation of small GTPases of the Ras superfamily by guanine nucleotide exchange factors (GEFs) is a key step in numerous cell signaling processes. Unveiling the detailed molecular mechanisms of GEF-GTPase signaling interactions is of great importance due to their central roles in cell biology, including critical disease states, and their potential as therapeutic targets. Here we present an assay to monitor individual Ras activation events catalyzed by single molecules of the GEF Son of Sevenless (SOS) in the natural membrane environment. The assay employs zero-mode waveguide (ZMW) nanostructures containing a single Ras-functionalized liposome. The ZMWs facilitate highly localized excitation of fluorophores in the vicinity of the liposome membrane, allowing direct observation of individual Ras activation events as single SOS enzymes catalyze exchange of unlabeled nucleotides bound to Ras with fluorescently labeled nucleotides from solution. The system is compatible with continuous recording of long sequences of individual enzymatic turnover events over hour-long time scales. The single turnover waiting time sequence is a molecular footprint that details the temporal characteristics of the system. Data reported here reveal long-lived activity states that correspond to well-defined conformers of SOS at the membrane. Liposome functionalized ZMWs allow for studies of nucleotide exchange reactions at single GTPase resolution, providing a platform to gauge the mechanisms of these processes.

SUBMITTER: Christensen SM

PROVIDER: S-EPMC5515077 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides.

Christensen Sune M SM Triplet Meredith G MG Rhodes Christopher C Iwig Jeffrey S JS Tu Hsiung-Lin HL Stamou Dimitrios D Groves Jay T JT

Nano letters 20160330 4

Activation of small GTPases of the Ras superfamily by guanine nucleotide exchange factors (GEFs) is a key step in numerous cell signaling processes. Unveiling the detailed molecular mechanisms of GEF-GTPase signaling interactions is of great importance due to their central roles in cell biology, including critical disease states, and their potential as therapeutic targets. Here we present an assay to monitor individual Ras activation events catalyzed by single molecules of the GEF Son of Sevenle ...[more]

PMID: 27013033

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Project description:An optical impedance spectroscopy (OIS) technique based on a single-mode electro-active-integrated optical waveguide (EA-IOW) was developed to investigate electron-transfer processes of redox adsorbates. A highly sensitive single-mode EA-IOW device was used to optically follow the time-dependent faradaic current originated from a submonolayer of cytochrome c undergoing redox exchanges driven by a harmonic modulation of the electric potential at several dc bias potentials and at several frequencies. To properly retrieve the faradaic current density from the ac-modulated optical signal, we introduce here a mathematical formalism that (i) accounts for intrinsic changes that invariably occur in the optical baseline of the EA-IOW device during potential modulation and (ii) provides accurate results for the electro-chemical parameters. We are able to optically reconstruct the faradaic current density profile against the dc bias potential in the working electrode, identify the formal potential, and determine the energy-width of the electron-transfer process. In addition, by combining the optically reconstructed faradaic signal with simple electrical measurements of impedance across the whole electrochemical cell and the capacitance of the electric double-layer, we are able to determine the time-constant connected to the redox reaction of the adsorbed protein assembly. For cytochrome c directly immobilized onto the indium tin oxide (ITO) surface, we measured a reaction rate constant of 26.5 s(-1). Finally, we calculate the charge-transfer resistance and pseudocapacitance associated with the electron-transfer process and show that the frequency dependence of the redox reaction of the protein submonolayer follows as expected the electrical equivalent of an RC-series admittance diagram. Above all, we show here that OIS with single-mode EA-IOW's provide strong analytical signals that can be readily monitored even for small surface-densities of species involved in the redox process (e.g., fmol/cm(2), 0.1% of a full protein monolayer). This experimental approach, when combined with the analytical formalism described here, brings additional sensitivity, accuracy, and simplicity to electro-chemical analysis and is expected to become a useful tool in investigations of redox processes.

Dataset Information

Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides.

Publications

Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides.

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