Biochemistry

Divalent metal ion transporters are conserved across all domains of life and play essential roles in diverse processes such as manganese acquisition during nutritional immunity in bacteria and iron homeostasis in higher eukaryotes [1–3]. Traditional techniques, such as electrophysiological assays, are often unsuitable due to the slow kinetics of many membrane transporters, electroneutral nature of certain transporter types, and the influence of other proteins with similar activity. To overcome these limitations and to investigate both the activity and ion selectivity of transporters, also including those normally expressed intracellularly, we have developed a fluorescence-based transport assay using purified proteins. This in vitro assay uses encapsulated fluorophores to monitor the movement of divalent metal ions (e.g., Mn²⁺, Ca²⁺, Mg²⁺) or protons across liposomal membranes reconstituted with purified transporter proteins. This approach provides detailed functional insight that complements structural and cellular data.

Enzyme immobilization offers a number of advantages that improve biocatalysis; however, finding a proper way to immobilize enzymes is often a challenging task. Implanting enzymes in metal–organic frameworks (MOFs) via co-crystallization, also known as biomineralization, provides enhanced reusability and stability with minimal perturbation and substrate selectivity to the enzyme. Currently, there are limited metal–ligand combinations with a proper protocol guiding the experimental procedures. We have recently explored 10 combinations that allow custom immobilization of enzymes according to enzyme stability and activity in different metals/ligands. Here, as a follow-up of that work, we present a protocol for how to carry out custom immobilization of enzymes using the available combinations of metal ions and ligands. Detailed procedures to prepare metal ions, ligands, and enzymes for their co-crystallization, together with characterization and assessment, are discussed. Precautions for each experimental step and result analysis are highlighted as well. This protocol is important for enzyme immobilization in various research and industrial fields.

Key features

• A wide selection of metal ions and ligands allows for the immobilization of enzymes in metal–organic frameworks (MOFs) via co-crystallization.

• Step-by-step enzyme immobilization procedure via co-crystallization of metal ions, organic linkers, and enzymes.

• Practical considerations and experimental conditions to synthesize the enzyme@MOF biocomposites are discussed.

• The demonstrated method can be generalized to immobilize other enzymes and find other metal ion/ligand combinations to form MOFs in water and host enzymes.

Graphical overview

This protocol describes a method for detecting and quantifying calcium ions in the endoplasmic reticulum (ER) and cytoplasm of cultured cells using fluorescent reporter proteins and ImageJ software. Genetically engineered fluorescent reporter proteins, such as G-CEPIA1er and GCaMP6f, localize to intracellular regions of interest (i.e., ER and cytoplasm) and emit green fluorescence upon binding to calcium ions. In this way, the fluorescence brightness of cells transfected with expression vectors for these reporters reflects the calcium ion concentration in each intracellular region. Here, we describe procedures for observing cultured cells expressing these fluorescent reporters under a fluorescence microscope, analyzing the obtained image using the free image analysis software ImageJ (https://imagej.net/ij/index.html), and determining the average fluorescence brightness of multiple cells present in the image. The current method allows us to quickly and easily quantify calcium ions on an image containing multiple cells and to determine whether there are relative differences in intracellular calcium ion concentration among experiments with different conditions.

Key features

• Detection and quantification of calcium ions in the ER and cytoplasm using fluorescent reporter proteins

• Quick and easy verification of measurement results using ImageJ

• Simultaneous comparison between various experimental conditions (drug treatment, mutants, etc.)

Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion composition. Here, we describe a calcein quenching assay based on large unilamellar vesicles that we used to evaluate divalent cation transport of the ionophore 4-Br-A23187. This assay can be used to study metal transport by ionophores and membrane proteins, under well-defined conditions.

Graphical abstract: