Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes
Key Takeaways
This 2009 paper serves as the origin story of Labbot. Faced with the need to characterize the response of a pH sensitive probe with very high precision, Thom decided to automate the task. The device featured a CCD detector with the capacity to automatically record absorbance spectra as a set of computer controlled syringe pumps added small volumes of acid or base.It was clear already from the beginning that this method not only reduced the hassle of making these measurements, but also greatly improved the data density, quality and precision.
Abstract
Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu3, which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe’s pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe’s pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu3 was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu3 was found to be superior to the commercially available pH indicators.
Other Publications Featuring Labbot
Modulating protein unfolding and refolding via the synergistic association of an anionic and a nonionic surfactant
This paper explores the complex interactions between proteins and surfactants. Through careful titration, the authors reveal how proteins undergo unfolding and refolding at varying ratios of ionic and nonionic surfactants, uncovering unexpected behaviors along the way. A deeper understanding of this interplay is essential for applications that rely on surfactants to stabilize proteins, including drug formulation, biotechnology, and beyond.
Insulin Amyloid Morphology is Encoded in H-bonds and Electrostatics Interactions Ruling Protein Phase Separation
This article investigates how hydrogen bonds and electrostatic interactions between ions and proteins influence phase separation and amyloid formation. Using a combination of microscopy, small-angle X-ray scattering, and ThT fluorescence, the authors map out how different ions on the Hofmeister scale alter the aggregation landscape.
Disassembly and in vitro cell compatibility of α-lactalbumin particulates under physiologically relevant conditions
The study showcases excellent techniques and controls for accurately measuring conditions inside droplets. Additionally, it offers insights into molecular tools for fine-tuning the critical concentration of synthetic condensates and modulating the partitioning of small molecules within them.