Integral membrane proteins (IMPs) are vital components in biology, carrying out essential functions in cells. Studying their structures is crucial for understanding their roles and for developing new medicines, as many drugs target these proteins. However, IMPs are notoriously difficult to work with because they don’t dissolve well in water, the usual medium for biological experiments. This is where detergents come in, helping to extract IMPs from their native environments. Yet, traditional detergents can sometimes damage or destabilize these delicate proteins. This article delves into a promising new class of molecules called GNG amphiphiles – but What Does Gng Mean and why are they important?
Understanding GNG: Glucose-Neopentyl Glycol Amphiphiles
The acronym GNG stands for Glucose-Neopentyl Glycol. GNG amphiphiles are a novel type of surfactant specifically designed for manipulating membrane proteins. Surfactants, like detergents, have both water-loving (hydrophilic) and water-fearing (hydrophobic) parts. This dual nature allows them to interact with both water and the fatty, lipid environment of cell membranes, making them useful for extracting and studying IMPs.
GNG amphiphiles are structurally similar to another successful class called MNG (maltose-neopentyl glycol) amphiphiles. However, a key difference lies in the sugar head group: GNG amphiphiles use glucose, while MNG amphiphiles use maltose. This seemingly small change can lead to significant differences in how they interact with membrane proteins, much like the differences observed between common detergents like OG (n-octyl-β-D-glucopyranoside) and DDM (N-Dodecyl-β-D-maltoside).
The development of GNG amphiphiles was motivated by the need for tools that can form small protein-detergent complexes (PDCs). Smaller PDCs are often advantageous for crystallizing membrane proteins, a critical step in determining their high-resolution structures using X-ray diffraction. While detergents like DDM are good at stabilizing membrane proteins, they tend to form larger PDCs, which can hinder crystallization. On the other hand, detergents forming smaller PDCs, such as OG and LDAO (lauryldimethylamine-N-oxide), can sometimes compromise protein stability. GNG amphiphiles were designed to bridge this gap, aiming for small PDC formation without sacrificing protein stability.
GNG Amphiphile Structures and Properties
As depicted in Figure 1, GNG amphiphiles feature a hydrophilic region composed of two glucose units and a hydrophobic region with two alkyl chains. Different GNG amphiphiles vary in how these alkyl chains are linked to the central neopentyl glycol core. For instance, GNG-1 and GNG-2 have ether linkages, while GNG-3 and GNG-4 have direct connections. Among these, GNG-4 exhibited poor water solubility and was not investigated further. GNG-1, GNG-2, and GNG-3, however, are highly water-soluble.
Researchers evaluated key properties of GNG amphiphiles, including their critical micelle concentration (CMC) – the concentration above which they start to form micelles (aggregates of surfactant molecules) – and the hydrodynamic radius (Rh) of these micelles. Compared to conventional glucose-based detergents like OG and OTG, GNG amphiphiles generally have lower CMC values, indicating they are more efficient at forming micelles. Micelle size analysis revealed that GNG-1 and GNG-3 form micelles smaller than DDM but larger than OG, while GNG-2 forms larger micelles than DDM. This variation in micelle size is crucial as it can influence the size of the PDCs formed with membrane proteins.
Evaluating GNG Amphiphiles with Membrane Proteins
To assess the effectiveness of GNG amphiphiles, researchers tested them on several different membrane protein systems, comparing their performance to that of traditional detergents like DDM, OG, and LDAO.
1. Photosynthetic Superassembly (LHI-RC) from Rhodobacter capsulatus
The first test involved the photosynthetic superassembly LHI-RC, a complex from Rhodobacter capsulatus that is known to be sensitive to detergents, especially the labile light-harvesting complex I (LHI). The stability of this superassembly when solubilized with GNG amphiphiles was compared to that with conventional detergents. Remarkably, the superassembly maintained its stability for 20 days when solubilized with either GNG-1 or GNG-2, performing as well as DDM. In contrast, using LDAO or OG led to rapid decomposition of the superassembly. This indicated that GNG amphiphiles, particularly GNG-1 and GNG-2, could effectively stabilize this delicate membrane protein complex.
2. Leucine Transporter (LeuT) from Aquifex aeolicus
Next, the leucine transporter LeuT, a different type of membrane protein from Aquifex aeolicus, was examined. Unlike the photosynthetic superassembly, LeuT showed a different response to GNG amphiphiles. GNG-1, GNG-2, and GNG-3 were found to be less effective than DDM in maintaining LeuT’s activity over 12 days. Among the GNG amphiphiles, GNG-2 showed the best performance, while GNG-1 and GNG-3 resulted in faster activity decline. This highlights that the optimal surfactant can vary depending on the specific membrane protein being studied.
Stability time course of (a) R. capsulatus LHI-RC photosynthetic superassembly and (b) LeuT, showing the performance of GNG-1 and GNG-2 compared to conventional detergents.
3. Succinate: Quinone Oxidoreductase (SQR) and Rhomboid Protease GlpG
To further broaden the evaluation, two more prokaryotic membrane proteins, SQR and GlpG, were tested. Thermostability assays revealed that all three GNG amphiphiles (GNG-1, GNG-2, and GNG-3) were slightly more effective than DDM in preserving the native state of both SQR and GlpG.
Detailed analysis of GlpG using gel filtration provided further insights. After incubation at 30°C, DDM-solubilized GlpG showed signs of aggregation and degradation. In contrast, GNG-2-solubilized GlpG showed no aggregation and minimal degradation. Furthermore, the GNG-2-GlpG complex eluted at a higher retention volume than the DDM-GlpG complex in gel filtration, indicating that GNG-2 forms smaller PDCs with GlpG compared to DDM. Molecular weight estimations confirmed that GlpG remained monomeric in both DDM and GNG-2, but the overall PDC size was smaller with GNG-2.
4. Membrane Protein Extraction
The ability of GNG-2, the most promising GNG amphiphile, to extract membrane proteins from biological membranes was also assessed. Studies using the R. capsulatus LHI-RC superassembly, LeuT, and CMP-Sia-GFP fusion protein showed that GNG-2 was generally comparable to DDM in its extraction efficiency.
GNG-3 and Membrane Protein Crystallization
The potential of GNG amphiphiles in promoting membrane protein crystallization was highlighted by the successful crystallization of the E. coli acetate transporter using GNG-3. Although the initial crystals diffracted to 4.1-Å resolution, this result was encouraging. Crucially, subsequent research by Kellosalo and colleagues achieved a high-resolution (2.6-Å) crystal structure of a sodium-pumping pyrophosphatase using GNG-3. This landmark achievement represents the first successful high-resolution structure determination of an IMP with a novel surfactant from the GNG family, underscoring the potential of GNG amphiphiles in advancing membrane protein structural biology.
Conclusion: The Significance of GNG Amphiphiles
In conclusion, GNG amphiphiles represent a valuable addition to the toolkit for membrane protein research. They exhibit a unique profile, effectively solubilizing and stabilizing various membrane proteins while tending to form smaller protein-detergent complexes. This combination of properties suggests that GNG amphiphiles, particularly GNG-3, hold significant promise for membrane protein crystallization, potentially complementing the strengths of MNG amphiphiles and traditional detergents.
While MNG amphiphiles generally excel in protein stabilization, mirroring the stabilizing effect of DDM compared to OG, GNG amphiphiles, like OG, appear to be more conducive to forming smaller PDCs. This characteristic makes them particularly interesting for PDC-based crystallization approaches, whereas MNG amphiphiles may be more suited for LCP (lipid cubic phase)-based crystallization. The development and characterization of GNG amphiphiles provide researchers with new options for tackling the challenges of membrane protein structural determination, ultimately furthering our understanding of these critical biological molecules.
References
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(Supplementary Material, Acknowledgements, Footnotes, Contributor Information, Notes and references, Associated Data sections can be adapted from the original article, ensuring all links and information are correctly updated if needed.)
