School of Science Division of Life Science 17 Cryo-EM Study of Membrane Proteins Supervisor: DANG Shangyu / LIFS Student: LIU Hangyu / BIBU Course: UROP 1100, Fall UROP 2100, Spring In the last report, we explored the protein family of sodium ion/proton exchangers (NHEs). By control analysis of the NHA2 proteins homologous to the NHA1, we got some insight into the NHA1. And through a series of expression and purification experiments, we got the general structure of the NHA1. In this report, we will further explore the structure of the functions of the NHA1. Firstly, we study what will happen if the protein is in sodium-free conditions and pH variability to observe its conformation changes and examine the environment’s influence on its conformation and functions. Secondly, by altering specific residues on this protein, examine residues’ functions to the protein. Thirdly, utilizing molecular dynamics simulations to comprehensively analyze the whole process of the dynamics of conformation changes. To Validate whether Any1 is a Retromer Cargo Supervisor: GAO Jieqiong / LIFS Student: GALAPPATHTHI Inushi Dinethsa / BIOT-AB Course: UROP 1000, Summer Any1 is a protein isolated from Saccharomyces cerevisiae that is believed to localise to endosomes and transGolgi network membranes. Its function is hypothesised to coordinate with Vps13 at Endoplasmic Reticulumendosome contact sites to aid endosome expansion by scrambling lipids between the endosome membrane bilayer as a phospholipid scramblase (Gao et al., 2025). However, Any1’s structure is yet to be experimentally determined, and its predicted structure via AlphoFold2 reveals that it is a transmembrane protein with a topology similar to that of PQ-loop proteins. To investigate this further, the Any1 protein was purified using an optimised protein purification protocol for structural elucidation via cryo-EM, and its function was analysed through a lipid scramblase assay. To Validate whether Any1 is a Retromer Cargo Supervisor: GAO Jieqiong / LIFS Student: LIU Yueyi / BCB Course: UROP 1000, Summer Protein purification is a fundamental process for determining the structure and regulation of proteins within cells. This process primarily involves protein extraction, purification techniques, and analytical approaches. Our research specifically targets Any1, which is a lipid scramblase in yeast cells (Gao et al, 2025). Identifying its structure would help discover the transportation and regulation of lipids between endosomes and lipids droplets in yeast cells. To determine its structure, we employ optimized purification methods based on protein nature predicted by Alphafold3. Here, we report a refined purification protocol for Saccharomyces cerevisiae phospholipid scramblase Any1, including reduction optimization of SDS-PAGE analysis, and detergent screening.
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