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Published in final edited form as: Curr Opin Struct Biol. 2025 Aug 6;94:103130. doi: 10.1016/j.sbi.2025.103130

Advances in Native Cell Membrane Nanoparticles System

Weihua Qiu a,b, Youzhong Guo a,b,*
PMCID: PMC12374768  NIHMSID: NIHMS2098884  PMID: 40774150

Abstract

The Native Cell Membrane Nanoparticles (NCMN) system utilizes membrane-active polymers specifically designed and optimized to extract and stabilize membrane proteins in the form of native cell membrane nanoparticles for biochemical and biophysical characterization. The NCMN system is a genuine and advanced detergent-free approach inspired by the membrane activity of the styrene-maleic acid co-polymers, distinguishing it from the Nanodisc technology, Salipro technology, and Peptidisc technology. The current advancements in the NCMN system, including the development of NCMN polymers, the application of the NCMN system for single-particle cryo-EM analysis, and the functional characterization of membrane proteins, are introduced in this review.

Keywords: Membrane protein, Cryo-EM, NCMN system, Structural biology, Drug discovery

Protein-lipid interactions and the natural structure and function of membrane proteins

Membrane proteins, located in the cell membrane system, play numerous crucial roles in normal physiology and are implicated in various pathological conditions; therefore, they also serve as primary targets for drugs. Both structural and functional information of membrane proteins are essential for understanding the active mechanism or developing better medicines; however, efforts to obtain this information are significantly hindered, essentially because membrane proteins pose notorious challenges to work with in vitro due to the lipid environment they rely on for their natural structure and function 1,2. Traditionally, in vitro characterization of membrane proteins depends on small-molecule detergents; however, besides annular lipids, some non-annular lipids are often removed from the transmembrane domain during the extraction and purification process in the presence of detergents 3. Membrane proteins, particularly those with multiple helices, are noteworthy; the cell membrane environment significantly influences their folding, natural structure, and function. Similarly, membrane proteins from the same protein family in different species may acquire new functions or exhibit distinct protein-lipid interactions for the same function 4. Several excellent reviews have discussed the importance of protein-lipid interactions, and the disruption of these natural protein-lipid interactions can lead to inaccurate structure and even a misleading interpretation of the active mechanism 3–13. The most recent and exceptional example demonstrating the essentiality of protein-lipid interactions is the case of the structure-enzyme activity relationships of particular methane monooxygenase (pMMO). Detergent-purified pMMO has no or minimal methane oxidation enzyme activity. In contrast, nanodisc-reconstituted pMMO could only partially but not fully restore the enzyme activity compared to the native cell membrane-associated pMMO 14,15.

Beginning of single particle cryo-EM analysis of membrane protein in native cell membrane lipid environments

In 2009, styrene maleic acid copolymer was first demonstrated to isolate membrane proteins from the cell membrane as nanoparticles 16. In 2018, the Styrene-Maleic Acid Lipid Particle (SMALP) platform was reported for the sub-nm single-particle cryo-EM structure determination of the E. coli AcrB 17; however, the resolution reached only 8.8 Å, and therefore, no structural information on protein-lipid interactions was obtained. Shortly after, the prototype of the Native Cell Membrane Nanoparticles system (NCMN system) was used for high-resolution cryo-EM structural analysis of E. coli AcrB, which revealed the well-ordered lipid bilayer structure within the transmembrane domain 18. The asymmetrically native lipid-associated AcrB structures differ remarkably from previously reported symmetric or pseudo-symmetric structures 19, 20, 21. Furthermore, it was shown that detergent application resulted in a loss of the natural lipid patch within the transmembrane domain, which could lead to significant conformational changes of the transmembrane domain upon a single mutation D407A 19; however, this is not the case in the form of native cell membrane nanoparticles 18. In the same year, high-resolution structures of Alternative complex III associated with native cell membrane lipids were also determined with the SMA co-polymer 22. The independent structure determination efforts using SMA copolymers marked the beginning of the single-particle cryo-EM analysis of membrane proteins in their native lipid environments. The prototypes of membrane-active polymers, such as SMA2000, SMA3000 and DIBMA, have significant limitations in their compatibility with low pH conditions and divalent cations 3, 23. Since then, numerous new SMA derivatives and other membrane-active polymers have been developed to overcome these limitations in general membrane protein research 24–38. However, the developed polymers have rarely been successfully used for high-resolution structure determination, and only a few high-resolution membrane protein structures have been reported, primarily using SMA copolymers 38. Furthermore, although high-resolution structures were sometimes solved using these polymers, surprisingly, protein-lipid interaction information could not be obtained 39, 40. Considering this situation, a more robust technology development is needed. It was initially known that SMA polymers have limitations in compatibility with low pH conditions and divalent ions; however, another significant limitation was that SMA polymers could also interfere with the transmembrane domain and damage the membrane protein structure 41. Sometimes, the disruption of the transmembrane domain by SMA or DIMBA co-polymers was even more severe than that of small-molecule detergents 3, 41, 42. In an earlier review article, Guo suggested that the advanced membrane-active polymers need to meet the following two standards for structural biology 3: 1) suitable for maintaining membrane proteins in the same functional states as on the native cell membranes; 2) suitable for high-resolution structure determination and retaining the structural information of essentially natural protein-lipid interactions.

Native cell membrane nanoparticles system and current advancement

Following the two standards, Guo and colleagues have focused on developing the Native Cell Membrane Nanoparticle system (Figure 1) 3, 43–49. Distinct from SMALP and other detergent-free or detergent-based approaches, the native cell membrane nanoparticles system emphasizes the natural protein-lipid interactions. For example, human membrane proteins should be expressed in human cells, but not in insect cells or bacterial cells, because the lipid environments do not match those of the membrane proteins. The native cell membrane nanoparticles system comprises three components: 1) NCMN polymer library. 2) Protocols for making the NCMN particles of membrane proteins. 3) Analytical approaches for characterization of NCMN particles, including but not limited to cryo-EM analysis, NMR analysis, X-ray crystallographic analysis, Mass Spectrometry analysis and various functional assays for transporters, channels, receptors and enzymes. In this mini-review, the current technological developments of the NCMN system will be introduced, covering NCMN polymer development, the application of the NCMN system for single-particle cryo-EM analysis of membrane proteins, and biochemical and biophysical characterizations of membrane proteins in the form of NCMN particles. Finally, the perspectives of the NCMN system in membrane protein structure biology and drug discovery will be briefly discussed.

Figure 1. Native cell membrane nanoparticles system (NCMN system).

Figure 1.

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The NCMN system comprises three components: A: NCMN polymers for stabilizing the membrane proteins; B: optimized NCMN protocols for preparation of NCMN particles; C: customized protocols for various structural and functional characterization of the membrane proteins in the form of NCMN particles.

NCMN polymer development

From prototype membrane-active polymers to the NCMNP-x-y series library

Prototype membrane-active polymers refer to completely hydrolyzed commercially available Styrene Maleic Anhydride (SMA) co-polymers such as SMA 1000, SMA 2000, SMA 3000, SMA 1440, SMA 2021, SMA 17352 from Cray Valley, and Diisobutylene Maleic Acids (DIBMA) from BASF (Sokalan® CP 9) and others. Among them, SMA2000 (NCMNP1–1 as indexed in the NCMN polymer library) is the most popular one (Figure 2A); however, the prototype membrane-active polymers including SMA2000 have significant limitations 3, 41, 50: 1) They are not compatible with lower pH conditions and usually only work well in solubilized cell membranes at the pH value above 7; 2) They are not compatible with divalent cations, such as Mg2+, Ca2+; 3) They did not work well for membrane proteins with flexible transmembrane domain in terms of high-resolution cryo-EM analysis; 4) They did not work well to maintain the enzyme activities of mammalian membrane protein enzymes.

Figure 2. General structures of representative NCMN polymers.

Figure 2.

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A. NCMNP1–1; B. NCMNP2a-x; C. NCMNP7-x; D. NCMNP13-x; E. NCMNP20-x; F. NCMNP21b-x.

We develop the first-generation NCMN polymers by fine-tuning the chemical and physical properties of the prototype membrane-active polymers to enhance the compatibility of the NCMN polymer series with divalent ions and low pH conditions. We also ensure their suitability for high-resolution structure determination and functional characterizations of membrane proteins.

NCMNP2a-x series.

To overcome the limitations of SMA co-polymers and investigate the potential influence of reducing carboxylic acid groups on polymer properties and subsequent membrane protein solubilization, we developed a novel series of NCMNP2a-x co-polymers (Figure 2B) 43. The NCMNP2a-x series polymers exhibit increased compatibility in lower pH conditions: NCMNP2a-5 is compatible with pH 5, NCMNP2a-25 is compatible with pH 4.0, NCMNP2a-50 is compatible with pH 3.0, and NCMNP2a-70 is compatible with pH 2.0. While the compatibility with the pH range has expanded, the NCMNP2a-x polymers do not exhibit significant compatibility with divalent ions, such as calcium. NCMNP2a-5, 25, and 50 are suitable for solubilizing a membrane protein for high-resolution structure determination. The high-resolution cryo-EM structure of E. coli AcrB, with associated native cell membrane lipids, was successfully determined. This new structure is superior to the one solved with SMA2000, as previously reported. SMA2000 may have distorted one of the alpha helices (1α helix), which is parallel to the cell membrane. Compared to the SMA 2000, the NCMNP2a-x polymers stabilize the structure of the more flexible region, such as the outer helix (Iα helix), effectively (Figure 3) 43. Furthermore, the TSPO enzyme activity assay demonstrated that NCMNP2a-50 is suitable for characterizing membrane proteins under lower pH conditions, specifically at pH levels of 3–4 43. Membrane-active polymers compatible with low pH conditions are necessary for characterizing membrane proteins in acidic environments, such as those found in locally acidic cellular compartments, including lysosomes and mitochondria.

Figure 3. Local structural differences of E. coli AcrB resulted from using SMA2000 and NCMNP2a-x.

Figure 3.

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The structural differences in local regions are red-outlined. A. AcrB structure missing the Iα helix resulted from using SMA2000 (PDB: 6BAJ) 18; B. AcrB structure resulted from using NCMNP2a-50 (PDB: 7RR7) 43; C. AcrB structure resulted from using NCMNP2a-5 (PDB: 7RR8) 43.

NCMNP7 series.

To overcome the divalent cation compatibility issue and maintain pH compatibility, we developed the NCMNP7-x series polymer (Figure 2C). The difference between NCMNP7-x and NCMNP2a is the grafted functional group. Instead of using Tris in the case of NCMNP2a-x, we grafted taurine to the carboxyl group in the case of NCMNP7-x (Figures 2B and C). The resulting NCMNP7-x is more compatible with lower pH conditions and divalent ions. It is also suitable for isolating and purifying membrane proteins, as well as for subsequent high-resolution cryo-EM analysis of various membrane proteins 45, 47, 51, 52.

MCMNP13-x, NCMNP21-x, and NCMNP21b-x series and others.

To explore the structure-property relationship, we also designed MCMNP13-x, NCMNP21-x, and NCMNP21b-x series polymers (Figures 2D, E, and F) 44. The results show that all polymers could solubilize the model membrane proteins. However, the morphologies of the NCMN particles differ significantly. For example, NCMNP13–50 and NCMNP21b-20 produced nice homogeneous single particles; however, NCMNP21–20 produced amorphous precipitates, and NCMMP21b-30 produced a mixture of homogeneous single particles and large lipid bilayer patches. The structure-property relationship analysis suggests that NCMNP21b-x polymers have vastly improved compatibility with divalent ions and a wider pH range, making them suitable for membrane protein structural biology. Furthermore, we have also developed other NCMN polymers, which have been collected into the first generation of the NCMN polymer library. Some are patent pending 49, 53, 54.

The NCMN system for structural biology and structure-based drug design

Initially, the prototype NCMN system was used to solve a high-resolution single-particle cryo-EM structure of AcrB associated with native cell membrane lipids 18. The lipid bilayer patch within the transmembrane domain formed a well-organized structure. Since then, multiple other membrane protein structures, including transporters, channels, enzymes, and receptors from bacteria and humans, have also been determined using the NCMN system (to be published separately). Here, the structure determination of human integrin demonstrates the NCMN system’s suitability for high-resolution cryo-EM analysis of human membrane proteins and structure-based drug design. Integrins are the primary receptors that animal cells employ to establish a connection with the extracellular matrix. They are heterodimers that are transmembrane linkers between the actin cytoskeleton and the extracellular matrix. Obtaining a high-resolution full-length integrin structure is very challenging. Collaborating with Amin Arnaout and colleagues, the NCMN system was used to successfully solve the first full-length, high-resolution cryo-EM structure of an integrin family member, specifically the human platelet integrin αIIbβ3 (Figure 4) 51, 52. It is worth noting that the integrin αIIbβ3 was purified from a natural source, the blood platelet membrane, without the use of any affinity tag. This case aligns well with the philosophy behind the native cell membrane nanoparticles system, namely, creating cell membrane nanoparticles from natural cells. Furthermore, physiologically functional integrin αIIbβ3 complexes need the presence of Mg2+ and Ca2+. The cryo-EM structure reveals the binding of Mg2+ and Ca2+ to the integrin, demonstrating that the NCMN system is superior to the prototype of membrane-active polymers, such as SMA2000, in its higher compatibility with divalent cations (Figure 3B) 52. Recently, using the NCMN system, we also solved the high-resolution cryo-EM structure of integrins αIIbβ3 with an improved antagonist, m-tirofiban (Figures 4B and C) 52. This suggests the NCMN system could potentially be used for structure-based drug discovery.

Figure 4. Full-length cryo-EM structure of human integrin αIIβ3 in NCMN particles.

Figure 4.

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A. Full-length apo-state integrin αIIβ3 structure (PDB: 8T2V); B. Enlarged m-Tirofiban (Cyan color) coordinates with a Mg2+ (Green colored sphere) binding in the integrin αIIβ3 structure; C. Full-length integrin αIIβ3 complexed with m-Tirofiban (PDB: 9DEQ).

The NCMN system for other biochemical and biophysical characterizations of membrane proteins

Besides applications for single-particle cryo-EM analysis of a broad range of membrane proteins from bacteria to humans, the NCMN system has also been successfully used for calcium and pH-dependent membrane protein enzyme activity assay 45, proteoliosome reconstitution for functional assay of membrane protein channels 46, Membrane protein drug target and ligand binding assay 47, and membrane protein-protein interaction analysis 48. Finally, it is worth noting that the NCMN system has also contributed to the discovery of the cholesterol-dependent protoporphyrin-IX oxygenase activity of human mitochondrial TSPO 50.

Perspectives of the NCMN system

The rapid development of cryo-EM technology, mainly single-particle cryo-EM and cryo-EM tomography, has significantly accelerated the advance of membrane protein structural biology 55, 56. However, we still have a significant bottleneck: sample preparation. Detergent-based approaches and derivatives, including nanodisc 57, peptidisc 58, and salipro 59 technologies, will continue to contribute to our understanding of membrane protein structure and function; however, because of the nature of the involvement of the detergents, we have to be cautious with the potential damage to the natural protein-lipid interactions, as recently demonstrated in the case of pMMO 14, 15. Once the natural protein-lipid interactions are damaged, it is challenging to restore the genuine interactions, even with natural cell membrane lipids reconstituted in nanodiscs or salipro particles 3, 14, 15. The NCMN system aims to prepare high-quality membrane protein samples for single-particle cryo-EM analysis and cryo-EM tomographic analysis in native cell membrane lipid environments. At the current stage of technology development of the NCMN system, we have developed an initial NCMN polymer library that is generally compatible with a broad range of pH conditions, divalent cations, and suitable for high-resolution cryo-EM analysis and functional characterization of membrane proteins from both prokaryotic and eukaryotic cells including some challenging human membrane proteins, such as integrins. However, this is only a promising beginning; there are still many challenges that need to be overcome. For example, 1) Some challenging membrane protein families with dynamic transmembrane domains, such as mechanosensitive channels, remain resistant to forming homogeneous NCMN particles, which hinders high-resolution cryo-EM structural analysis. 2) The crystallization of the NCMN particles has not been successful yet, which limits current structural analysis of the NCMN particles to single-particle cryo-EM. 3) Proteoliposome reconstitution with NCMN particles remains inefficient because a detergent-free environment is not ideal for the fusion of the membrane protein with preformed liposomes. 4) Accurate lipid assignment to the Cryo-EM density remains a challenge; sometimes, due to flexibility, the shape of the density is often inadequate to assign specific lipids. To overcome these challenges, new NCMN polymers need to be developed, and protocols for manufacturing NCMN particles need to be experimentally optimized. The development of membrane-active polymers could be facilitated through rational design, following the accumulation of experimentally validated membrane-active polymers. Furthermore, various creative biochemical and biophysical characterization approaches need to be developed to cater to the detergent-free system.

It is necessary to comment on the current development of the structure-predicting tools. AlphaFold and other evolving structure prediction tools will be helpful to experimental structural biologists 60. However, experimental structures will remain essential for accurately interpreting the structure-activity relationship of membrane proteins. Currently, accurate structural information on protein-lipid interactions remains scarce. AlphaFold and other structure prediction tools cannot reliably predict membrane protein structures for basic research or drug discovery practices without an accurate and comprehensive structure database for training. The NCMN system and other genuine detergent-free systems, including SMALP technology 16, cell-derived membrane vesicles 61, and in situ cryo-EM tomography 62, may play essential roles in advancing the biology of membrane proteins and drug discovery related to them.

Acknowledgments

NIH R01-GM132329 to Y.G. supports work on the NCMN system in the Guo laboratory. The VCU School of Pharmacy, Department of Medicinal Chemistry, and the Center for Drug Discovery, formerly known as the Institute for Structural Biology, Drug Discovery, and Development, also support the Guo laboratory through its provision of laboratory space and facilities.

Footnotes

Declaration of competing interests

Y.G. and W.Q. have patents pending for the NCMN system through Virginia Commonwealth University. Y.G. and W.Q. are founders of NCMNtech LLC, a startup company.

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Papers of particular interest, published within the period review, have been highlighted as:

* of special interest

** of outstanding interest.

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