



The CHIANG LAB
- Publications
- …
- Publications



The CHIANG LAB
- Publications
- …
- Publications

Membrane proteins by ESR
Detergents vs. membrane vesicles vs. nanodisc
Membrane proteins are integral to numerous cellular processes, yet their conformational dynamics in native environments remain difficult to study. This study introduces a nano-delivery method using nanodiscs to transport spin-labeled membrane proteins into the membranes of living cells, enabling direct in-cell double electron-electron resonance (DEER) spectroscopy measurements. We investigated the membrane protein BsYetJ, incorporating spin labels at key positions to monitor conformational changes. Our findings demonstrate successful delivery and high-quality DEER data for BsYetJ in both Gram-negative E. coli and Gram-positive B. subtilis membranes. The delivered BsYetJ retains its ability to transport calcium ions. DEER analysis reveals distinct conformational states of BsYetJ in different membrane environments, highlighting the influence of lipid composition on protein structure. This nano-delivery method overcomes traditional limitations, enabling the study of membrane proteins in more physiologically relevant conditions.
The 18-kDa translocator protein (TSPO) has gained considerable attention as a clinical biomarker for neuroinflammation and a potential therapeutic target. However, the mechanisms by which TSPO associates with ligands, particularly the endogenous porphyrin ligand, protoporphyrin IX (PpIX), remain poorly understood. In this study, we employed mutagenesis and spectroscopy-based functional assays to investigate TSPO-mediated photo-oxidative degradation of PpIX and identify key residues involved in the reaction. We provide structural evidence using electron spin resonance, which sheds light on the highly conserved intracellular loop (LP1) connecting transmembrane 1 (TM1) and TM2. Our findings show that LP1 does not act as a lid to regulate ligand binding; instead, it interacts strongly with the TM3-TM4 linker (LP3) to stabilize the local structure of LP3. This LP1-LP3 interaction is crucial for maintaining the binding pocket structure, which is essential for proper ligand binding. Our results also demonstrate that PpIX accesses the pocket through the lipid bilayer without requiring conformational changes in TSPO. This study provides an improved understanding of TSPO-mediated PpIX degradation, highlighting potential therapeutic strategies to regulate the reaction.
Structure and regulation of the BsYetJ calcium channel
The endoplasmic reticulum (ER) serves as the major intracellular calcium store. The release of calcium from this store regulates a variety of cellular functions, hence playing a key role in regulating apoptosis. The transmembrane BAX inhibitor-1 motif-containing 6 (TMBIM6) is an evolutionarily conserved multifunctional ER membrane protein that mediates calcium homeostasis to protect against apoptosis and ER stress. We study a bacterial homolog of TMBIM6 and report several previously unidentified conformational substates involved in the calcium transport process. Our findings stimulate a model of calcium-release channel showing that membrane protein functioning requires the interconversion between distinct conformations in preexisting conformational equilibria.
ESR reveals lipid-dependent conformational changes of membrane proteins
Membrane proteins are incorporated in various environments, including detergent, membrane vesicles, and nanodisc. ESR distance measurements show that the protein behave differently with environments.
Chiang Lab@NTHU