Imaging Ca²⁺ Concentration and pH in Nanopores/Channels of Protein Crystals

Protein crystals are nanoporous materials. Despite this important characteristic, little is known about the conditions in the pores, also called channels. Here, we describe a method to study the calcium concentration and pH in the nanopores of thaumatin and lysozyme crystals. We load the crystal nanopores with fluorescent indicators and then perfuse the crystals with solutions of different calcium concentrations and pH while reading out the crystal's fluorescence intensity with confocal microscopy. By calibrating the fluorescence signal, we can determine the calcium concentration and pH in the nanopores. For the pH in thaumatin nanopores measured with the ratiometric pH sensor SNARF-1, we find a -0.7 pH shift compared to the bath pH corresponding to a fivefold higher proton concentration. This is similar to the -0.3 pH shift found in lysozyme nanopores. With single-wavelength probes, we find that the calcium concentration in thaumatin crystal nanopores is the same as in the bath, whereas it is 0.24 times lower in lysozyme nanopores. Summarizing, our experiments show that calcium concentration and pH in the nanopores of protein crystals can deviate significantly from that in the bath. In general, the described method can be applied for testing a wide range of ion or small-molecule concentrations in transparent nanoporous materials not only with ratiometric but also with single wavelength fluorescent indicators.