The regulation of pH is essential for proper organelle function, and organelle-specific changes in pH often reflect the dynamics of physiological signaling and metabolism. For example, mitochondrial energy production depends on the proton gradient maintained between the alkaline mitochondrial matrix and neutral cytosol. However, we still lack a quantitative understanding of how pH dynamics are coupled between compartments and how pH gradients are regulated at organelle boundaries. Genetically encoded pH sensors are well suited to address this problem because they can be targeted to specific subcellular locations and they facilitate live, single-cell analysis. However, most of these pH sensors are derivatives of green and yellow fluorescent proteins that are not spectrally compatible for dual-compartment imaging. Therefore, there is a need for ratiometric red fluorescent protein pH sensors that enable quantitative multicolor imaging of spatially resolved pH dynamics. In this work, we demonstrate that the I158E/Q160A mutant of the red fluorescent protein mCherry is an effective ratiometric pH sensor. It has a pKa of 7.3 and a greater than 3-fold change in ratio signal. To demonstrate its utility in cells, we measured activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells. Furthermore, we were able to image pH changes simultaneously in the cytosol and mitochondria by using the mCherryEA mutant together with the green fluorescent pH sensor, ratiometric-pHluorin. Our results demonstrate the feasibility of studying interorganelle pH dynamics in live cells over time and the broad applicability of these sensors in studying the role of pH regulation in metabolism and signaling.

Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first report the 1.09 Å X-ray crystal structure of mCherryTYG, exhibiting a fully matured chromophore. We next determine that it has an extraordinarily large dynamic range with a 2 nanosecond lifetime change from pH 5.5 to pH 9.0. Critically, we find that the sensor maintains a pKa of 6.8 independent of environment, whether as the purified protein in solution or expressed in live cells. Furthermore, the lifetime measurements are ro-bustly independent of total fluorescence intensity and scatter. We demonstrate that mCherryTYG is a highly effective sensor using time resolved fluorescence spectroscopy on live-cell suspensions, which has been previously overlooked as an easily accessible approach for quantifying intracellular pH. As a red fluorescent *

Matrix metalloproteinases (MMPs) are crucial proteases in maintaining the health and integrity of many tissues, however their dysregulation often facilitates disease progression. In disease states these remodeling and repair functions support, for example, metastasis of cancer by both loosening the matrix around tumors to enable cellular invasion and by affecting proliferation and apoptosis, and they promote degradation of biological restorations by weakening the substrate to which the restoration is attached. As such, MMPs are important therapeutic targets. MMP-8 participates in cancer, arthritis, asthma and failure of dental fillings. MMP-8 differs from other MMPs in that it has an insertion that enlarges its active site. To elucidate the unique features of MMP-8 and develop selective inhibitors to this therapeutic target, a stable and active form of the enzyme is needed. MMP-8 has been difficult to express at high yield in a soluble, active form. Typically recombinant MMPs accumulate in inclusion bodies and complex methods are applied to refold and purify protein in acceptable yield. Presented here is a streamlined approach to produce in E. coli a soluble, active, stable MMP-8 fusion protein in high yield. This fusion shows much greater retention of activity when stored refrigerated without glycerol. A variant of this construct that contains the metal binding claMP Tag was also examined to demonstrate the ability to use this tag with a metalloprotein. SDS-PAGE, densitometry, mass spectrometry, circular dichroism spectroscopy and an activity assay were used to analyze the chemical integrity and function of the enzyme.

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pH regulation plays a crucial role in protein function, cell metabolism, intracellular degradation, and a wide range of other physiological processes. Monitoring perturbations in local pH environments due to cellular activities or diseases, such as increased neuron activity, receptor trafficking, or extracellular acidosis in a tumor microenvironment, would better our understanding of disease pathology. Live‐cell imaging with fluorescent protein‐based sensors allows us to monitor broad pH changes; however, there are currently limited options to quantitatively measure pH dynamics with subcellular resolution in live specimens. Concentration and intensity dependence of the fluorophore, sample thickness, and photo‐bleaching are limiting factors, and additionally, most of the current genetically encoded pH sensors are generated from green fluorescent protein, limiting our ability to perform multiplex imaging to monitor pH simultaneously in different compartments. As an intrinsic property of fluorophores, fluorescence lifetime does not rely on protein concentration, method of measurement or fluorescence intensity. Utilizing this property, we can monitor discrete compartmental changes in pH using fluorescence lifetime imaging microscopy (FLIM), while opening up a wider selection of potential fluorescent sensors for multi‐channel imaging. It is the goal of this project to develop genetically‐encoded red fluorescent pH sensors to quantitatively measure micro‐environmental pH changes using fluorescence lifetime. Here we present in vitro and live‐cell characterization; in proof‐of‐concept studies we have utilized FLIM to measure the pH of different subcellular compartments simultaneously in real‐time. Our results demonstrate our progress towards understanding the nuances of subcellular pH and aberrant cell behavior, and how the role of pH is affected in both healthy and disease states.Support or Funding InformationEmily Haynes is supported by her Charles H. Viol Chemistry Fellowship. We also gratefully acknowledge support from the Showalter Fund and from National Institutes of Health grants R21 NS092010 and R21 EY026425.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.