Membrane proteins hold a pivotal position in cellular functionality, overseeing critical processes encompassing signal transduction, transport mechanisms, and cellular adhesion. Nevertheless, their intrinsic hydrophobic nature introduces substantial complexities in the realms of isolation and characterization. Conventional techniques traditionally rely on the utilization of detergents to solubilize these proteins, a methodology fraught with challenges. Detergents, while efficient in their solubilization efforts, often introduce harsh conditions that can result in protein denaturation or aggregation. Consequently, the imperative for alternative, detergent-free approaches are essential for membrane protein purification and reconstitution.
Local Lipid Polymer-Nanodiscs
In the quest to address the limitations posed by conventional detergents, scientists have embarked on a journey of innovation, finding solace in novel solutions, such as local lipid polymer-nanodiscs. These nanodiscs provide a biocompatible environment for membrane proteins by incorporating them into a lipid bilayer enclosed by amphipathic polymers. The solubilization of cell membranes and the isolation of membrane proteins within these nanodiscs offer several advantages.
First and foremost, the employment of local lipid polymer-nanodiscs creates a native-like environment for membrane proteins. This environment plays a pivotal role in safeguarding the structural integrity and optimal functionality of these proteins—a paramount prerequisite for conducting precise investigations into their biochemical attributes and intricate biological roles. Additionally, these nanodiscs are highly versatile and can be tailored to mimic the specific lipid composition of different cell membranes. This flexibility allows scientists to study a wide range of membrane proteins from various cellular sources.
Fig 1. Schematic showing the formation of polymer-nanodiscs upon mixing synthetic lipids/membranes (liposomes; yellow/blue) with an amphipathic polymer (green). (Krishnarjuna B, Ramamoorthy A. 2022)
Characterization of Local Lipid Polymer-Nanodiscs
Characterizing local lipid polymer-nanodiscs stands as a pivotal and intricate step in the multifaceted realm of membrane protein studies. This pursuit relies on a spectrum of specialized techniques, notably dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopy, which wield the power to unveil the size and composition of the nanodiscs. Moreover, mass spectrometry can help verify the incorporation of the target membrane protein into the nanodiscs.
Table 1. List of membrane proteins reconstituted in nanodiscs and studied by NMR spectroscopy. (Krishnarjuna B, Ramamoorthy A. 2022)
| Protein(s) Reconstituted |
Nanodisc Type |
| Human cytochrome P450 3A4 (CYP3A4) |
MSP |
| Human voltage-dependent anion channel-1 (VDAC-1) |
MSP |
| Human voltage-dependent anion channel-2 (VDAC-2) |
MSP |
| VDAC N-terminal segment (NTS) |
MSP |
| The transmembrane domain of stromal interaction molecule (STIM1-TM) |
MSP |
| Bacterial β-barrel assembly machinery-A (BamA) |
MSP |
| Bacteriorhodopsin |
MSP |
| Outer membrane protein X (OmpX) |
MSP |
| α–helical BLT2 G protein-coupled receptor |
MSP |
| NTS8–13–NTSR1–Gαi1β1γ1 complex |
MSP |
| hIAPP |
MSP |
| Anti-apoptotic protein BclxL |
MSP |
| Inner mitochondrial MPV17 |
MSP |
| Bak transmembrane helix |
MSP |
| Y. pestis Omp adhesion invasion locus (Ail) |
MSP |
| Human interleukin-8 (IL-8)-CXCR1(1–38) complex |
MSP |
| Rabbit cytochrome-b5 + horse cytochrome C |
4F peptide |
| Rabbit CYP450 2B4, rat CYP450 reductase FMN-binding domain, and rabbit cytochrome-b5 |
4F peptide |
| Cytochrome-b5 + CYP450 |
22A peptide |
| Pf1, p7 from human hepatitis C virus and human chemokine receptor CXCR1 (GPCR) |
18A peptide |
| Rabbit cytochrome-b5 |
SMA-EA |
| Pf1 coat protein |
SMA |
| MerFt, CXCR1 and Ail |
SMA |
| Rat CYP450 reductase FMN-binding domain |
Pentyl-inulin |
Amphipathic Polymers
Local lipid polymer-nanodiscs have enabled scientists to isolate a wide range of membrane proteins from various cell membranes. From G-protein coupled receptors (GPCRs) to ion channels and transporters, these nanodiscs have revolutionized our ability to study the functions and structures of diverse membrane proteins.
Amphipathic polymers, such as styrene maleic acid (SMA) copolymers, have been particularly instrumental in this regard. SMA copolymers have a unique amphipathic nature, allowing them to interact with both hydrophobic membrane regions and aqueous environments. This property makes them ideal for extracting and isolating membrane proteins without the need for harsh detergents.
High-Resolution Structure Determination Using Cryo-EM
Cryo-electron microscopy (cryo-EM) has emerged as an invaluable tool in the realm of structural biology, particularly in elucidating the intricate three-dimensional configurations of membrane proteins nestled within nanodiscs. With its capability to achieve near-atomic resolution, cryo-EM has ushered in a new era of scientific discovery.
This groundbreaking technique has propelled our comprehension of membrane protein structures to unprecedented heights, unraveling the subtle nuances of their folding, dynamic conformational changes, and intricate interactions with ligands. These revelations hold immense promise, illuminating novel avenues for drug development, especially in the realm of therapeutics targeting membrane proteins, which play pivotal roles in a spectrum of diseases.
Reference
- Krishnarjuna B, Ramamoorthy A. Detergent-Free Isolation of Membrane Proteins and Strategies to Study Them in a Near-Native Membrane Environment. Biomolecules. 2022; 12(8):1076.
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