Jan L. Vinkenborg

Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic β cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles.

Enhanced sensitivity of FRET-based protease sensors by redesign of the GFP dimerization interface

changes in a sensor domain are translated into a change in energy-transfer efficiency between donor and acceptor fluorescent domains, which is detected by a change in the ratio of donor and acceptor emission. This ratiometric response is independent of the sensor concentration, which is an important advantage of FRET-based sensors. In practice, however, most FRET-based sensors display only a relatively small difference in emission ratio upon activation. Improvement of these ratiometric changes has been recognized as an important prerequisite for use of these sensor systems in high-throughput applications based on fluorescence plate readers and fluorescence assisted cell sorting (FACS). [14, 15] Recently a pair of CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) variants, CyPet and YPet, respectively, have been reported that were optimized for FRET through a process of directed evolution. [16] When incorporated in a protease sensor, a 20-fold change in emission ratio was observed upon cleavage of a flexible peptide that linked CyPet and YPet, compared to only a fourfold change for the same construct with enhanced CFP (ECFP) and enhanced YFP (EYFP) domains. However, the mechanism behind their remarkable FRET properties has remained unclear. A total of eighteen mutations were introduced in the course of their development, many of which were at the exterior of the protein, at a large distance from the fluorophore. Moreover, no large differences in quantum yield or extinction coefficient were reported; this suggests that the photophysical properties of the fluorescent proteins were not significantly altered. We therefore hypothesized that the increase in FRET observed for CyPet and YPet could be due to an enhanced tendency to interact when connected by a peptide linker. The parent green fluorescent protein (GFP) has a known tendency to dimerize, [17] and analysis of the mutations in YPet have identified two residues, S208F and V224L, that are present at the dimer interface, as shown by the X-ray structure of the GFP dimer. Here, we show that A of just these two mutations in both fluorescent domains of ECFP–linker–EYFP constructs results in a fourfold increase in the EYFP-to-ECFP emission ratio, which yields a 16fold change in emission ratio upon protease cleavage of the peptide linker (Figure 1). Additional biophysical evidence is provided, which shows that the mutations indeed result in A of an intramolecular complex.

Fluorescent imaging of transition metal homeostasis using genetically encoded sensors.

The ability to image the concentration of transition metals in living cells in real time is important for understanding transition metal (TM) homeostasis and its involvement in diseases. Genetically encoded fluorescent sensor proteins are attractive because they do not require cell-invasive procedures, can be targeted to different locations in the cell, and allow ratiometric detection. Important progress in the development of Zn(2+) sensors has allowed sensitive detection of the very low free concentrations of Zn(2+) in single cells, both in the cytosol and various organelles. Together with other recent advances in chemical biology, these tools seem particularly useful to interrogate the dynamics and compartmentation of TM homeostasis.

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation.

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Engineering protein switches: sensors, regulators, and spare parts for biology and biotechnology.

Switch protein switch! Proteins that switch between distinct conformational states are ideal for monitoring and controlling molecular processes in biological systems. We discuss new engineering concepts for the construction of protein switches that have the potential to be generally applicable and discuss them according to their mechanism of action.

MagFRET : the first genetically encoded fluorescent Mg2+ sensor

Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg2+, MagFRET-1. This sensor is based on the high-affinity Mg2+ binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg2+-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg2+ affinity (K d = 148 µM) with a 50% increase in emission ratio upon Mg2+ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg2+ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg2+ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg2+ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca2+, this affinity is sufficiently attenuated (K d of 10 µM) to make the sensor insensitive to known Ca2+ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg2+ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg2+ sensors could prove very useful in understanding intracellular Mg2+ homeostasis and signaling.

Rational design of FRET sensor proteins based on mutually exclusive domain interactions

Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio. The modularity of this approach should make it easily transferable to other applications of protein switches in fields ranging from synthetic biology, optogenetics and molecular diagnostics.