Addressing the challenges in membrane protein characterisation

Dr Sofia Ferreira, Director of Applications at Refeyn, discusses how various technologies can address the main challenges in membrane protein characterisation, describing how novel technologies, such as mass photometry, can streamline and support analytical processes. 

Dissecting the structure of membrane proteins 

Membrane proteins play a crucial role in cellular functions, serving as essential components for intercellular communication, catalysing vital energy transformations, and facilitating the transport of molecules in and out of cells and intracellular compartments. Given their role in regulating cellular functions, it comes as no surprise that membrane proteins are the primary targets for the majority of currently marketed therapeutics. 

However, while membrane proteins constitute approximately 20–30% of gene coding proteins1, integral membrane proteins such as transporters, ion channels, and GPCRs remain under-represented in the protein data bank (the PDB), a global repository for structural 2. This is largely due to their long amino acid sequences and their hydrophobic surfaces, which makes experimentally determining their structures, and thereby their function, incredibly challenging. Consequently, there is a demand for advanced technologies that are better able to resolve and characterize complex hydrophobic proteins.  

Without structural and functional information available, it is challenging to develop new drugs that can target relevant integral membrane proteins.  

Challenges in membrane protein characterization 

To understand the function of membrane proteins, it’s essential to have information on their three-dimensional structure. Membrane proteins can be analysed using multiple techniques such as X-ray crystallography, single-particle cryo-electron microscopy (Cryo-EM), and receptor–ligand binding assays. However, these biophysical methods are all but impossible to conduct in the native environment and so require the protein to be extracted from the membrane and studied in a detergent or lipid environment in vitro. 

While this sounds simple enough, attempting to extract membrane proteins from their native environment often results in pronounced structural and functional ramifications3,4. Scientists use a multitude of membrane mimetics to overcome this issue. They experimentally study membrane proteins using detergents, nanodiscs, styrene maleic acid lipid particles (SMALPs), and amphiphilic polymers (amphipols) to purify and stabilise fully functional membrane proteins. However, mimicking the membrane can be extremely difficult as several factors, including lipid composition, membrane curvature, asymmetry, tension, and fluidity, can directly impact the structural and functional integrity of membrane proteins.  

As such, when using these membrane mimetics, it’s crucial to identify the optimal conditions to prevent MP damage and preserve function and structure, which can be an extremely difficult and time-consuming process. Consequently, the selection and optimisation of membrane mimetics is one of the main bottlenecks in structural and functional studies of membrane proteins. 

Understanding the bottleneck 

Traditionally, proteins are extracted from their native environment using synthetic detergents designed to mimic and replace the lipids that normally surround the protein. Detergents are often used to solubilise and purify membrane proteins because of their comparative ease of use, making it possible to efficiently extract proteins directly from isolated membranes or intact cells. However, detergents only provide a very basic imitation of the native lipid bilayer and have been shown to cause destabilisation and denaturation of some proteins, in addition to increasing sample heterogeneity5. 

Given the huge variety of detergents currently available, the identification of a detergent that fulfills specific protein requirements, in terms of stability and functionality, can be a painstaking trial and error process3. 

Many novel technologies have been developed to overcome the limitations of detergent-based approaches, including nanodiscs, amphipols, and SMALPs (Fig 1). These membrane-mimetic systems (MMS) are able to replicate the composition of the biological cell membrane, offering a more accurate representation than detergent-based methods. Though useful, these MMS still come with certain caveats, such as a narrow range of stable temperatures and pHs, limited diversity of detergent or lipid types, poor control over sample homogeneity, and effects on MP integrity and function. This means that their use still requires multiple purification and optimisation steps, usually in the form of ‘trial and error’ screening, which is time-consuming and sample-intensive6 

Figure 1. A visual representation of the different membrane mimetic systems that are used in membrane protein characterization workflows.

Given the distinct requirements of different MMS, careful purification and characterization of MP preparations are crucial for both functional and structural research. Current methods for assessing MP stability involve chromatography and SDS-PAGE, followed by further analysis with size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), multi-angle light scattering (MALS), Nano differential scanning fluorimetry (NanoDSF), negative stain electron microscopy (EM), or native mass spectrometry (MS). 

While these processes are well-established, they are also time-consuming and often challenging. 

For example, MALS characterization demands a significant amount of material and can only quantitatively analyse well-resolved peaks. Similarly, AUC also requires substantial volume of samples and several hours to analyse a small number, while native MS requires a high level of expertise and specialised equipment.  

EM is an established method for the determination of 3D structural information for a wide range of biomolecules. However, it is also known for being a time-consuming process that demands careful optimisation of staining conditions and sample quality. Moreover, this technique requires specialised expertise in order to acquire reliable data and perform subsequent analysis. On the other hand, SEC offers a more accessible option for sample analysis, but typically delivers lower resolution compared to other advanced techniques. 

NanoDSF is a relatively new technology that is fast, easy to use, and uses very little material. However, it does depend on proteins containing aromatic amino acids (tryptophan or tyrosine) that change their local chemical environment upon denaturation.  

Given these challenges and caveats, a method that offers detailed, rapid, and accurate sample characterization in solution could significantly enhance and accelerate structural and functional in vitro studies of membrane proteins. 

How can we streamline this process? 

Mass photometry, a relatively recent bioanalytical technique, has proven to be an effective means of overcoming the bottlenecks associated with membrane protein characterization. As a single-molecule technique, it provides high-resolution information on the heterogeneity of a sample, and is able to produce results within minutes. 

By quantifying the light scattered by individual molecules in solution, mass photometry can provide insight into the molecular mass, purity, and behaviour of membrane proteins, including crucial aspects like aggregation, oligomerization, and interaction dynamics (Fig 2). This approach enables researchers to rapidly and accurately measure the effects of different mimetic types and conditions on the state of their proteins of interest. 

Figure 2. The principle of mass photometry. The light scattered by a molecule that has landed on a measurement surface interferes with light reflected by that surface. The interference signal scales linearly with mass.

As a result, mass photometry is emerging as a powerful tool in the field of MP research, playing a key role in characterizing membrane proteins using detergent systems8, amphipols9, nanodiscs10, and SMALPs. Additionally, the method has been shown to deliver similar results to other well-established techniques, such as SEC-MALS and AUC. Table 1 shows a detailed comparison of various methods used to assess membrane protein stability.

Optimising membrane protein experiments  

Despite their drawbacks, detergents continue to be widely used for extracting and purifying membrane proteins, making it essential to have efficient tools for analysing their impact on sample solubility and behaviour under different concentrations and buffer conditions. Mass photometry not only eliminates the necessity for complete detergent removal but also enables quick assessment of solubility conditions on a case-by-case basis. 

In a recent study on the fungal pathogen Ustilago maydis, researchers harnessed mass photometry to identify and characterize two membrane proteins implicated in the disease8. This technique was also crucial in assessing how protein concentrations and detergent types influenced their oligomerization. To address potential noise from high detergent concentrations, the researchers used a rapid in-drop dilution method to successfully minimise interference. Mass photometry played a key role in optimising the study and independently identifying and characterizing the target proteins. 

Table 1. A comparison of analytical technologies used for assessing membrane protein stability.

As with detergents, it is crucial to find the optimal conditions for MP experiments when using amphipols, SMALPs, and nanodiscs. In a 2021 study, researchers examined different methods to incorporate the KcsA potassium channel from Streptomyces lividans into nanodiscs10. 

Using SEC, the researchers found that two of these preparations exhibited nearly identical profiles. However, they were able to use mass photometry to reveal a crucial distinction: KcsA was only properly assembled into functional tetramers within the nanodiscs of one preparation. This was confirmed by functional analysis that showed that only the tetramer-containing preparation displayed protein activity. This demonstrated that the single-molecule sensitivity and resolution of mass photometry can provide critical insights for structural and functional nanodisc studies that may elude other techniques.  

The ongoing development of mass photometry, such as the integration of rapid dilution and microfluidics, is broadening the horizons of membrane protein characterization. These advancements confer the benefit of rapidly removing detergent-induced background noise and enabling measurements before membrane proteins precipitate12. As a result, a wider range of membrane proteins and diverse experimental conditions can be analysed swiftly and with greater accuracy. 

The power of collaboration 

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant to cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments.  

While MMS continue to improve, there is still a need for careful optimisation regardless of the system used. Current techniques for assessing membrane protein stability are very powerful and have enabled detailed information on sample composition and heterogeneity, but they are also extremely slow and require complex preparations and analysis. Mass photometry can serve as a complementary and supportive technology in this endeavor. It offers versatility, speed, and ease of use, facilitating a deeper understanding of these important cellular components and the development of novel therapeutics. By working in tandem with established methods, mass photometry can accelerate the progress in membrane protein research.  

About the author  

Dr Sofia Ferreira specialises in biochemistry and microbiology. During her PhD and postdoctoral research, Ferreira characterized autokinase, a membrane protein, studying its interactions and role in virulence, biofilm formation, and host-pathogen interactions. Transitioning to industry, she developed several assays within the medical devices industry and more recently in Refeyn, she has been exploring the full potential of mass photometry. 

 References: 

  1. Fischer, F., Wolters, D., Rögner, M., & Poetsch, A. (2006). Toward the Complete Membrane Proteome: High Coverage of Integral Membrane Proteins Through Transmembrane Peptide Detection. Molecular & Cellular Proteomics, 5(3), 444–453. https://doi.org/10.1074/mcp.M500234-MCP200  
  1. Li, Z., & Buck, M. (2021). Beyond history and “on a roll”: The list of the most well-studied human protein structures and overall trends in the protein data bank. Protein Science, 30(4), 745–760. https://doi.org/10.1002/pro.4038 
  1. Seddon, A. M., Curnow, P., & Booth, P. J. (2004). Membrane proteins, lipids and detergents: Not just a soap opera. Biochimica Et Biophysica Acta, 1666(1–2), 105–117. https://doi.org/10.1016/j.bbamem.2004.04.011  
  1. Chorev, D. S., & Robinson, C. V. (2020). The importance of the membrane for biophysical measurements. Nature Chemical Biology, 16(12), 1285–1292. https://doi.org/10.1038/s41589-020-0574-1  
  1. Yang, Z., Wang, C., Zhou, Q., An, J., Hildebrandt, E., et al. (2014). Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains. Protein Science : A Publication of the Protein Society, 23(6), 769–789. https://doi.org/10.1002/pro.2460  
  1. Young, J. W. (2023). Recent advances in membrane mimetics for membrane protein research. Biochemical Society Transactions, 51(3), 1405–1416. https://doi.org/10.1042/BST20230164  
  1. Chen, A., Majdinasab, E. J., Fiori, M. C., Liang, H., & Altenberg, G. A. (2020). Polymer-Encased Nanodiscs and Polymer Nanodiscs: New Platforms for Membrane Protein Research and Applications. Frontiers in Bioengineering and Biotechnology, 8. https://www.frontiersin.org/articles/10.3389/fbioe.2020.598450  
  1. Weiland, P., & Altegoer, F. (2021). Identification and Characterization of Two Transmembrane Proteins Required for Virulence of Ustilago maydis. Frontiers in Plant Science, 12. https://www.frontiersin.org/articles/10.3389/fpls.2021.669835  
  1. Webby, M. N., Oluwole, A. O., Pedebos, C., Inns, P. G., Olerinyova, A., et al. (2022). Lipids mediate supramolecular outer membrane protein assembly in bacteria. Science Advances, 8(44). https://doi.org/10.1126/sciadv.adc9566  
  1. Olerinyova, A., Sonn-Segev, A., Gault, J., Eichmann, C., Schimpf, J., et al. (2021). Mass Photometry of Membrane Proteins. Chem, 7(1), 224–236. https://doi.org/10.1016/j.chempr.2020.11.011  
  1. Beenken, A., Cerutti, G., Brasch, J., Guo, Y., Sheng, Z., et al. (2023). Structures of LRP2 reveal a molecular machine for endocytosis. Cell, 186(4), 821-836.e13. https://doi.org/10.1016/j.cell.2023.01.016  
  1. Refeyn. (2023). Mass photometry analysis of samples at micromolar concentration. Application Note. Retrieved from Refeyn website: https://info.refeyn.com/app-note-massfluidix  

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