Search

Gefördert im Rahmen des Projekts DEAL

Summary Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers—in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation., Graphical Abstract, Highlights • We introduce a label-free, single molecule approach for membrane-protein characterization • Mass photometry quantifies membrane proteins in different membrane-mimetic systems • MP reveals carrier and protein heterogeneity • It helps distinguish different functional states of membrane proteins, The Bigger Picture Membrane proteins are some of the most important biological molecules, carrying out vital functions and being frequent drug targets. Yet, preferring lipid environments and so requiring solubilization, they are challenging to study. Here, we show that mass photometry can characterize the heterogeneity of membrane proteins and the carriers in which they are solubilized. It can also distinguish different functional states of membrane proteins. Our approach thus opens the door to more comprehensive studies of function, structure, and interaction of these critical proteins in their native membrane environment at the single-molecule level., Membrane proteins are important in cell signaling and disease. They are also difficult to study as they require solubilization from lipids by membrane-mimetic systems. We show that mass photometry can facilitate the study of membrane proteins in various mimetic systems. With this method, we can distinguish different oligomeric and functional states of membrane proteins, opening the door for in vitro functional studies, structural characterization, and protein-protein interaction analysis of membrane proteins.

Size-exclusion chromatography coupled to multiangle laser light scattering (SEC-MALLS) is the perfect method to determine the oligomeric state of membrane proteins as this method works in solution and is totally independent from prior assumptions such as detergent-to-protein ratio or the shape of the protein. In a relatively short time (ca. 30 min), the molecular mass and quality of a membrane protein preparation can be determined. Here, I provide a detailed protocol on how to perform a SEC-MALLS run and show exemplary chromatograms and their analysis.

Correction for ‘Trehalose-cored amphiphiles for membrane protein stabilization: importance of the detergent micelle size in GPCR stability’ by Manabendra Das et al., Org. Biomol. Chem., 2019, 17, 3249–3257.

With the recent success in determining membrane protein structures, further detailed understanding of the identity and function of the bound lipidome is essential. Using an approach that combines high-energy native mass spectrometry (HE-nMS) and solution-phase lipid profiling, this protocol can be used to determine the identity of the endogenous lipids that directly interact with a protein. Furthermore, this method can identify systems in which such lipid binding has a major role in regulating the oligomeric assembly of membrane proteins. The protocol begins with recording of the native mass spectrum of the protein of interest, under successive delipidation conditions, to determine whether delipidation leads to disruption of the oligomeric state. Subsequently, we propose using a bipronged strategy: first, an HE-nMS platform is used that allows dissociation of the detergent micelle at the front end of the instrument. This allows for isolation of the protein-lipid complex at the quadrupole and successive fragmentation at the collision cell, which leads to identification of the bound lipid masses. Next, simultaneous coupling of this with in-solution LC-MS/MS-based identification of extracted lipids reveals the complete identity of the interacting lipidome that copurifies with the proteins. Assimilation of the results of these two sets of experiments divulges the complete identity of the set of lipids that directly interact with the membrane protein of interest, and can further delineate its role in maintaining the oligomeric state of the protein. The entire procedure takes 2 d to complete.

Background: Mitochondrial carriers were thought to be dimeric based on their migration in blue native gels. Results: The high molecular mass species observed in blue native gels are composed of protein monomers, detergent, lipid, and Coomassie stain. Conclusion: The mitochondrial carriers are monomeric not dimeric. Significance: The apparent mass of small membrane proteins in blue native gels requires significant correction., Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ∼32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from ∼60 to ∼130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ∼120 kDa, but appears smaller on gels (∼60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein.

1. Chlorfenapyr [4-bromo-2-(4-chlorophenyl)-1-(ethoxymethl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile] is a commonly employed pesticide throughout the world. The mechanism of chlorfenapyr action is to uncouple oxidative phosphorylation in the mitochondria. The characteristic features of chlorfenapyr intoxication are high fever, rhabdomyolysis and neurologic symptoms that gradually get worse until death. 2. In recent years, suicide attempt cases using commercial chlorfenapyr pesticide were reported. Even small doses of commercial chlorfenapyr pesticide intoxication caused human fatality. However, world health organization (WHO) has classified chlorfenapyr as class 2-moderately hazardous chemical. Animal studies using technical grade (94.5%; AC 7504-59A) chlorfenapyr in 0.5% carboxy methyl cellulose as the vehicle, single dose through oral route in male rats were well tolerated. 3. We planned a therapeutic strategy for suicidal chlorfenapyr intoxication, therefore we evaluated the three different toxic doses of chlorfenapyr (10% chlorfenapyr and 90% detergent) through oral route in male rats for human extrapolation. The major difference between the technical grade chlorfenapyr and commercial grade chlorfenapyr was the vehicle. In the technical grade chlorfenapyr study, 0.5% carboxy methyl cellulose was used as a vehicle, whereas in the present study 90% detergent acted as a vehicle. The LD50 of commercial grade chlorfenapyr-40.63 mg/kg bw, which was approximately tenfold decrease than technical grade chlorfenapyr, LD50 - 441 mg/kg bw. 4. The combination of chlorfenapyr and detergent, a deadly cocktail to form micelle complex that can greatly influence bioavailability by attaching to biological membranes in vivo. To conclude, the enhanced bioavailability of chlorfenapyr by the detergent causes the fatality in suicidal attempts using chlorfenapyr.

Mass spectrometry of intact membrane protein complexes requires removal of the detergent micelle by collisional activation. We demonstrate that the necessary energy can be obtained by adjusting the degree of collisional cooling in the ion source. This enables us to extend the energy regime for dissociation of membrane protein complexes.

Background: Methods for rapid assessment of interactions of small molecules with membrane proteins in detergent are lacking. Results: Thermostability measurements of mitochondrial transporters display informative trends about detergent, lipid, substrate, and inhibitor interactions. Conclusion: Mechanistic insights are obtained by studying the thermostability of mitochondrial transporters. Significance: Information about the nature of compound interactions with membrane proteins can be obtained rapidly., Mitochondrial carriers, including uncoupling proteins, are unstable in detergents, which hampers structural and mechanistic studies. To investigate carrier stability, we have purified ligand-free carriers and assessed their stability with a fluorescence-based thermostability assay that monitors protein unfolding with a thiol-reactive dye. We find that mitochondrial carriers from both mesophilic and thermophilic organisms exhibit poor stability in mild detergents, indicating that instability is inherent to the protein family. Trends in the thermostability of yeast ADP/ATP carrier AAC2 and ovine uncoupling protein UCP1 allow optimal conditions for stability in detergents to be established but also provide mechanistic insights into the interactions of lipids, substrates, and inhibitors with these proteins. Both proteins exhibit similar stability profiles across various detergents, where stability increases with the size of the associated detergent micelle. Detailed analysis shows that lipids stabilize carriers indirectly by increasing the associated detergent micelle size, but cardiolipin stabilizes by direct interactions as well. Cardiolipin reverses destabilizing effects of ADP and bongkrekic acid on AAC2 and enhances large stabilizing effects of carboxyatractyloside, revealing that this lipid interacts in the m-state and possibly other states of the transport cycle, despite being in a dynamic interface. Fatty acid activators destabilize UCP1 in a similar way, which can also be prevented by cardiolipin, indicating that they interact like transport substrates. Our controls show that carriers can be soluble but unfolded in some commonly used detergents, such as the zwitterionic Fos-choline-12, which emphasizes the need for simple validation assays like the one used here.

Application of Tris-N-[Tris(hydroxymethyl)methyl]glycine gels for 2DE is hampered by formation of mixed CHAPS-SDS micelles resulting in typical swirling pattern in the low mass range, which makes reliable quantitative and qualitative gel evaluation impossible. Modification of 2DE strip equilibration procedure prevented the direct interaction between both detergents during equilibration process, thus substantially improving gel separation.

The production of sufficient amounts of chemically and conformationally homogenous protein is a major requirement for successful crystallization and structure determination. With membrane proteins, this constitutes a particular problem because the membrane volume is limited and the organisms are usually very sensitive to changes in membrane properties brought about by massive protein insertion. Moreover, the extraction of membrane proteins from the membrane with detergents is generally a harsh treatment, which gives rise to conformational aberrations. A number of successful procedures for functional expression followed by purification are reviewed here together with nonfunctional expression into inclusion bodies and subsequent (re)folding to produce functional proteins. Most of the data are for prokaryotic outer membrane proteins, but the outer membrane proteins of eukaryotic organelles are also considered as they do show similar features.

Many attempts have been made to rationalize the use of detergents for membrane protein studies [J. Biol. Chem. 264 (1989) 4907]. The barrier properties of the detergent headgroup may be one parameter critically involved in protein protection. In this paper, we analyzed these properties using a model system, by comparing the accessibility of tryptophan octyl ester (TOE) to water-soluble collisional quenchers (iodide and acrylamide) in three detergent micelles. The detergents used differed only in the chemical nature of their polar headgroups, zwitterionic for dodecylphosphocholine (DPC) and nonionic for octa(ethylene glycol) dodecyl monoether (C(12)E(8)) and dodecylmaltoside (DM). In all cases, in phosphate buffer at pH 7.5, the binding of 5 microM TOE was complete in the presence of a slight excess of detergent micelles over TOE molecules, resulting in a significant blue shift and greater intensity of TOE fluorescence emission. The resulting quantum yield of bound TOE was between 0.08 (in DPC) and 0.12 (in DM) with an emission maximum (lambda(max)) of approximately 335 nm whatever the detergent micelle. Time-resolved fluorescence intensity decays of TOE at lambda(max) were heterogeneous in all micelles (3-4 lifetime populations), with mean lifetimes of 1.7 ns in DPC, and 2 ns in both C(12)E(8) and DM. TOE fluorescence quenching by iodide, in detergent micelles, yielded linear Stern-Volmer plots characteristic of a dynamic quenching process. The accessibility of TOE to this ion was the greatest with C(12)E(8), followed by DPC and finally DM (Stern-Volmer quenching constants K(sv) of 2 to 5.5 M(-1)). In contrast, the accessibility of TOE to acrylamide was greatest with DPC, followed by C(12)E(8) and finally DM (K(sv)=2.7-7.1 M(-1)). TOE also presents less rotational mobility in DM than in the other two detergents, as shown from anisotropy decay measurements. These results, together with previous TOE quenching measurements with brominated detergents [Biophys. J. 77 (1999) 3071] provide reference data for analyzing Trp characteristics in peptide (and more indirectly protein)-detergent complexes. The main finding of this study was that TOE was less accessible (to soluble quenchers) in DM than in DPC and C(12)E(8), the cohesion of DM headgroup region being suggested to play a role in the ability of this detergent to protect function and stability of solubilized membrane proteins.

The determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within ±15% to those reported previously, indicating a general applicability of this approach.

Knowledge of the energetic parameters of transmembrane helix–helix interactions is necessary for the establishment of a structure–energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix–helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain.