ZIP (Zrt/Irt-like protein) transporters, which regulate zinc (Zn 2+ ) homeostasis by importing Zn 2+ into cells, play a crucial role in regulating various biological processes in humans. These processes encompass gene transcription, enzymatic activity, and the immune response. Zn(II) dyshomeostasis can lead to diseases such as osteoarthritis, fatty liver disease, and various types of cancer emphasizing the importance of understanding ZIP transport mechanisms. Bb ZIP is hypothesized to function via an elevator-type movement, where its transport domain moves in parallel relative to the rigid scaffold domain. The motion is driven by the inherent voltage of the inner membrane, allowing for the alternating exposure of metal-binding sites on either side. To date, Bb ZIP has only been structurally studied in neutral voltage environments and adopts its inward-facing conformation. This research aims to determine the structural and functional dynamics of Bb ZIP by using a combination of time-resolved serial-femtosecond X-ray crystallography (TR-SFX), cryo-electron microscopy (cryo-EM), and proteoliposome reconstitution approaches to capture the elusive outward-facing conformation by exposing it to non-neutral voltage environments. Here, I present our work optimizing the expression and purification of Bb ZIP using a flag-tag purification methodology, which dramatically enhanced the yield and purity of isolated Bb ZIP. I also outline our experimental designs for future structural studies using TR-SFX and cryo-EM with proteoliposome reconstitution approaches.
Roy et al. (Sun,) studied this question.
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