Exploring the World of Membrane Proteins: Techniques and Methods for Understanding Structure, Function, and Dynamics
Abstract
:1. Introduction
2. Techniques and Methods Used for Protein Analysis
2.1. Separative Techniques
2.1.1. Electrophoresis
2.1.2. Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis or SDS-PAGE
2.2. Dimensional Electrophoresis (SDS-PAGE/Isoelectric Focusing)
2.2.1. Dimensional Electrophoresis (16-BAC/SDS-PAGE)
2.2.2. Blue Native PAGE
2.2.3. Capillary Electrophoresis
2.2.4. Free Flow Electrophoresis
2.3. Techniques for Characterization and Structural Analysis
2.3.1. X-ray Crystallography
2.3.2. Cryogenic Electron Microscopy
2.3.3. Nuclear Magnetic Resonance Spectroscopy
- Solution NMR is a technique that is important to study proteins in solution. It’s used to study membrane protein folding, interactions, conformational changes, and internal mobility, in addition to ligand-substrate interactions [124]. One of its main limitations is size, as it is particularly useful for studying small to medium-sized proteins. In the past decades, it went form only detecting 10 kDa proteins in the 1980s to around 25–35 kDa in the mid-1990s [117]. Recent advancements in high-field magnets and cryogenic probes, together with new sample preparation protocols and transverse relaxation-optimized methods, have pushed solution NMR protein size limitations to reach almost 100 kDa in some rare instances [117]. For instance, researchers were able to detect conformational changes in the CLC membrane transporter (100 kDa) by using a monomeric ClC-ec1 variant (50 kDa) [125]. Solution NMR has also contributed to the characterization of many integral membrane proteins [126]. These include Human voltage-dependent anion channel (VDAC-1) [127], Bacterial outer membrane protein G [128] and mitochondrial uncoupling protein 2 [129]
- Solid state NMR on the other hand, uses quick sample spinning or alignment to produce excellent resolution in membrane proteins [130]. One of the main areas where solid state NMR exceeds solution NMR is that ssNMR have no limitation on the size of the protein [131]. For instance, ssNMR has allowed the study of the structure and dynamic of BAM complex (200 kDa) in lipid bilayer [132].
2.4. Biophysical Techniques
2.4.1. Nanodiscs
2.4.2. Patch Clamp
2.4.3. Atomic Force Microscopy
2.4.4. Neutron Scattering
2.5. Computational Methods
3. Artificial Intelligence at the Service of Protein Structure
3.1. Application of AI
3.2. AI Methods in Biology
3.2.1. Alphafold2
3.2.2. RoseTTAFold
3.2.3. ESMFold
3.2.4. Improvements
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Separation and Analysis | ||||
---|---|---|---|---|
Technique | Description | Advantages | Limitations | References |
SDS-PAGE Sodium dodecyl-sulfate gel electrophoresis | Separation method allowing protein separation by mass |
|
| [27,28,29,59,60,61] |
2-Dimensional Electrophoresis (SDS-PAGE/IEF) | Technique combining SDS-PAGE and Isoelectric focusing for the separation based on pI and mass. |
|
| [34,35,62,63] |
2-Dimensional Electrophoresis (16-BAC/SDS-PAGE) | This combines SDS-PAGE and the use of the 16-BAC cationic detergent with a separation based on charge and hydrophobicity |
|
| [34,45,64,65] |
Blue Native PAGE (BN-PAGE) | While preserving proteins’ native state, this protocol is used to study and isolate membrane proteins. |
|
| [47,48,66,67,68,69] |
Capillary Electrophoresis | An analytical method separating charged proteins based on their electrical mobility. |
|
| [70,71,72,73,74] |
Free Flow Electrophoresis | This technique analyses a continuous stream of proteins on a channel with an electric field perpendicular to the flow. |
|
| [55,75,76,77] |
Characterization and Conformation | ||||
---|---|---|---|---|
Technique | Description | Advantages | Limitations | References |
Crystallography | Determines the structure of protein crystals using the diffraction patterns collected by X-rays, electrons, or neutrons. |
|
| [78,81,83,84,93,136] |
Cryogenic electron microscopy (Cryo-EM) | Visualizes high-resolution protein structures by imaging frozen samples with an electron microscope. |
|
| [96,101,103,104,137] |
Nuclear Magnetic Resonance (NMR) | Studies the nuclei in the atoms of protein to determine molecular structure, dynamics, and interactions. |
|
| [113,115,121,122,123,138] |
Biophysical Innovation | ||||
---|---|---|---|---|
Nanodiscs | Solubilizes membrane proteins in aqueous media while keeping them in a native-like environment. |
|
| [139,140,141] |
Patch clamp | Studies ion channels by studying the flow of ions through it. |
|
| [142,143,144] |
Atomic Force Microscopy (AFM) | Gives images and characterizes the surfaces of membrane proteins at the nanoscale by scanning a probe tip and measuring forces between the tip and sample. |
|
| [145,146,147,149,150,154,155] |
Neutron Scattering | Uses a beam of neutrons to determine the atomic structure, composition, dynamics, and magnetic properties of membrane proteins. |
|
| [151,156,157,158] |
Artificial Intelligence | ||||
---|---|---|---|---|
Technique | Description | Advantages | Limitations | References |
RoseTTAFold | “three-track” neural network developed by Baker lab, to predict the 3D structure of proteins from their amino acid sequences |
|
| [180] |
AlphaFold2 | Deep learning-based AI system developed by DeepMind that accurately predicts the 3D structure of proteins from their amino acid sequences |
|
| [178,184] |
AlphaFold-Multimer | An Alphafold model trained to predict protein-protein complexes |
|
| [181] |
ESMFold2 | AI system developed by meta that predicts protein structures using a large language model trained on a massive dataset of protein sequences. |
|
| [175] |
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Boulos, I.; Jabbour, J.; Khoury, S.; Mikhael, N.; Tishkova, V.; Candoni, N.; Ghadieh, H.E.; Veesler, S.; Bassim, Y.; Azar, S.; et al. Exploring the World of Membrane Proteins: Techniques and Methods for Understanding Structure, Function, and Dynamics. Molecules 2023, 28, 7176. https://doi.org/10.3390/molecules28207176
Boulos I, Jabbour J, Khoury S, Mikhael N, Tishkova V, Candoni N, Ghadieh HE, Veesler S, Bassim Y, Azar S, et al. Exploring the World of Membrane Proteins: Techniques and Methods for Understanding Structure, Function, and Dynamics. Molecules. 2023; 28(20):7176. https://doi.org/10.3390/molecules28207176
Chicago/Turabian StyleBoulos, Imad, Joy Jabbour, Serena Khoury, Nehme Mikhael, Victoria Tishkova, Nadine Candoni, Hilda E. Ghadieh, Stéphane Veesler, Youssef Bassim, Sami Azar, and et al. 2023. "Exploring the World of Membrane Proteins: Techniques and Methods for Understanding Structure, Function, and Dynamics" Molecules 28, no. 20: 7176. https://doi.org/10.3390/molecules28207176
APA StyleBoulos, I., Jabbour, J., Khoury, S., Mikhael, N., Tishkova, V., Candoni, N., Ghadieh, H. E., Veesler, S., Bassim, Y., Azar, S., & Harb, F. (2023). Exploring the World of Membrane Proteins: Techniques and Methods for Understanding Structure, Function, and Dynamics. Molecules, 28(20), 7176. https://doi.org/10.3390/molecules28207176