A colorimetric competitive displacement assay for the evaluation of catalytic peptides

An assay based on competitive dye displacement was adapted to detect the formation of aldol product in crude reaction mixtures, and was used to evaluate minimal peptide aldol catalysts.</p

The Taf14 YEATS domain is a reader of histone crotonylation

The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity

The Development of a Colorimetric Competitive Displacement Assay for the Evaluation of Catalytic Peptides

Synthetic peptides offer a rich source of robust and selective catalysts, given their inherent chirality and ease of synthesis. A limitation to exploring large sequence spaces is the evaluation of catalytic efficacy. We have developed a colorimetric competitive displacement assay to monitor reaction progress and, thus, evaluate the activity of potential catalysts for direct aldol reactions. The assay has been designed such that the aldol product displaces a dye from a receptor, resulting in a change in the absorbance of the dye that is used to monitor reaction progress. This assay provides an efficient colorimetric method for evaluating catalytic peptides

Measuring Absolute Membrane Potential Across Space and Time

Membrane potential (Vmem) is a fundamental biophysical signal present in all cells. Vmem signals range in time from milliseconds to days, and they span lengths from microns to centimeters. Vmem affects many cellular processes, ranging from neurotransmitter release to cell cycle control to tissue patterning. However, existing tools are not suitable for Vmem quantification in many of these areas. In this review, we outline the diverse biology of Vmem, drafting a wish list of features for a Vmem sensing platform. We then use these guidelines to discuss electrode-based and optical platforms for interrogating Vmem. On the one hand, electrode-based strategies exhibit excellent quantification but are most effective in short-term, cellular recordings. On the other hand, optical strategies provide easier access to diverse samples but generally only detect relative changes in Vmem. By combining the respective strengths of these technologies, recent advances in optical quantification of absolute Vmem enable new inquiries into Vmem biology

VoltageFluor dyes and fluorescence lifetime imaging for optical measurement of membrane potential

Membrane potential is a fundamental biophysical parameter common to all of cellular life. Traditional methods to measure membrane potential rely on electrodes, which are invasive and low-throughput. Optical methods to measure membrane potential are attractive because they have the potential to be less invasive and higher throughput than classic electrode based techniques. However, most optical measurements rely on changes in fluorescence intensity to detect changes in membrane potential. In this chapter, we discuss the use of fluorescence lifetime imaging microscopy (FLIM) and voltage-sensitive fluorophores (VoltageFluors, or VF dyes) to estimate the millivolt value of membrane potentials in living cells. We discuss theory, application, protocols, and shortcomings of this approach

Computationally Assisted Design of High Signal-to-Noise Photoinduced Electron Transfer-Based Voltage-Sensitive Dyes

High signal-to-noise optical voltage indicators will enable simultaneous interrogation of membrane potential in large ensembles of neurons. However, design principles for voltage sensors with high sensitivity and brightness remain elusive, limiting the applicability of voltage imaging. In this paper, we use molecular dynamics (MD) simulations and density functional theory (DFT) calculations to guide the design of a bright and sensitive green-fluorescent voltage-sensitive fluorophore, or VoltageFluor (VF dye), that uses photoinduced electron transfer (PeT) as a voltage-sensing mechanism. MD simulations predict an 11% increase in sensitivity due to membrane orientation, while DFT calculations predict an increase in fluorescence quantum yield, but a decrease in sensitivity due to a decrease in rate of PeT. We confirm these predictions by synthesizing a new VF dye and demonstrating that it displays the expected improvements by doubling the brightness and retaining similar sensitivity to prior VF dyes. Combining theoretical predictions and experimental validation has resulted in the synthesis of the highest signal-to-noise green VF dye to date. We use this new voltage indicator to monitor the electrophysiological maturation of human embryonic stem cell-derived medium spiny neurons. </p

Bioorthogonal, fluorogenic targeting of voltage-sensitive fluorophores for visualizing membrane potential dynamics in cellular organelles

Electrical potential differences across lipid bilayers play foundational roles in cellular physiology. Plasma membrane voltage is the most widely studied; however, the bilayers of organelles like mitochondria, lysosomes, nuclei, and endoplasmic reticulum (ER) also provide opportunities for ionic compartmentalization and the generation of transmembrane potentials. Unlike plasma membranes, organellar bilayers, cloistered within the cell, remain recalcitrant to traditional approaches like patch-clamp electrophysiology. To address the challenge of monitoring changes in organelle membrane potential, we describe the design, synthesis, and application of LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine based Voltage Reporter) for optically monitoring membrane potential changes in the endoplasmic reticulum (ER) of living cells. We pair a tetrazine-quenched RhoVR for voltage sensing with a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting to the ER. Bright fluorescence is observed only at the coincidence of LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of LUnAR RhoVR and Cer-TCO is voltage sensitive and that LUnAR RhoVR can be targeted to intact ER in living cells. Using LUnAR RhoVR, we use two-color, ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release from internal stores. Finally, we use LUnAR RhoVR to directly visualize functional coupling between plasma-ER membrane in patch clamped cell lines, providing the first direct evidence of the sign of the ER potential response to plasma membrane potential changes. We envision that LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology

Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles

Electrical potential differences across lipid bilayers play foundational roles in cellular physiology. Plasma membrane voltage is the most widely studied; however, the bilayers of organelles like mitochondria, lysosomes, nuclei, and the endoplasmic reticulum (ER) also provide opportunities for ionic compartmentalization and the generation of transmembrane potentials. Unlike plasma membranes, organellar bilayers, cloistered within the cell, remain recalcitrant to traditional approaches like patch-clamp electrophysiology. To address the challenge of monitoring changes in organelle membrane potential, we describe the design, synthesis, and application of the LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine-based Voltage Reporter) for optically monitoring membrane potential changes in the ER of living cells. We pair a tetrazine-quenched RhoVR for voltage sensing with a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting to the ER. Bright fluorescence is observed only at the coincidence of the LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of the LUnAR RhoVR and Cer-TCO is voltage-sensitive and that the LUnAR RhoVR can be targeted to an intact ER in living cells. Using the LUnAR RhoVR, we use two-color, ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release from internal stores. Finally, we use the LUnAR RhoVR to directly visualize functional coupling between the plasma-ER membranes in patch clamped cell lines, providing the first direct evidence of the sign of the ER potential response to plasma membrane potential changes. We envision that the LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology

The Taf14 YEATS domain is a reader of histone crotonylation

The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity