Two-dimensional electrophoresis of soybean root plasma membrane proteins solubilized by SDS and other detergents

Proteins solubilized from enriched soybean root plasma membrane with sodium dodecyl sulphate (SDS) and selected non-denaturing detergents (octyl-β-d-glucopyranoside, Zwittergent 312, Zwittergent 314, Zonyl FSK, and Nonidet P-40) were electrophoresed in two-dimensions by standard procedures. The basic electrophoretogram ‘fingerprint’ was similar for all detergents tested. However, differences in the total number of polypeptides resolved and the presence or absence of certain polypeptides on specific two-dimensional gels indicated some selectivity. Of all detergents tested, SDS solubilized the most polypeptides (ca 95) and provided the best resolution. The other detergents solubilized 50–80 polypeptides with varying resolution. Of those tested, octyl-β-d-glucopyranoside consistently provided the best balance between the number of polypeptides resolved (ca 70) and the level of resolution. The results suggest that selected detergents may prove useful in plant plasma membrane studies which require non-denaturing conditions.     

Electrofocusing of integral membrane proteins in mixtures of zwitterionic and nonionic detergents.

In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubilized proteins was preserved. A mixture of the zwitterionic detergent, SB₁₄, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing intergral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (pI range 7–11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of patter constancy, these conditions were achieved within 15 h, 0–4°C, at 200 V in 6-cm gels of 5% T/15% C_Bis with 0.1 n H₂SO₄ and 0.1 n KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.     

Stability and solubility of proteins from extremophiles

Charges are important for hyperthermophile protein structure and function. However, the number of charges and their predicted contributions to folded state stability are not correlated, implying that more charge does not imply greater stability. The charge properties that distinguish hyperthermophile proteins also differentiate psychrophile proteins from mesophile proteins, but in the opposite direction and to a smaller extent. We conclude that charge number relates to solubility, whereas protein stability is determined by charge location. Most other structural properties are poorly separated over the ambient temperature range, apart from the burial of certain amino acids. Of particular interest are large non-polar sidechains that tend to increased exposure in proteins evolved to function at higher temperatures. Looking at tryptophan in more detail, this increase is often located close to the termini of secondary structure elements, and is discussed in terms of a novel potential role in protein thermostabilisation.     

Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers

In the biosynthesis of the C7-cyclitol moiety, valienol, of the -glucosidase inhibitor acarbose in Actinoplanes sp. SE50/110 various cyclitol phosphates, such as 1-epi-valienol-7-phosphate, are postulated precursors. In the cell extracts of Actinoplanes SE50/110 we found a new kinase activity which specifically phosphorylates 1-epi-valienol; other C7-cyclitol analogs were only weakly or not phosphorylated. The purified product of the kinase reaction turned out to be 1-epi-valienol-7-phosphate in analyses by nuclear magnetic resonance spectroscopy. The enzyme seems not to be encoded by an acb gene and, therefore, plays a role in a salvage pathway rather than directly in the de novo biosynthesis of acarbose.     

Modifiable chromatophore proteins in photosynthetic bacteria.

The chromatophores of Chromatium vinosum, as well as six other photosynthetic bacteria, contained two or more proteins which were insoluble when heated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME). When the chromatophores were dissolved at room temperature in SDS-beta-ME, these proteins were present in the SDS-polyacrylamide gel electrophoresis profiles, but when the samples were dissolved at 100 degrees C, they were absent or considerably diminished. When one-dimensional gels of chromatophores solubilized at room temperature were soaked in the SDS-beta-ME solution and heated to 100 degrees C and the gels were run in a second dimension, the proteins became immobilized in the original first-dimension gel, where they could be detected by staining. The two major proteins so affected in C. vinosum had apparent molecular weights of 28,000 and 21,000. The chromatophores of several other photosynthetic bacteria also contained predominant proteins between 30,000 and 19,000 molecular weight, which became insoluble when heated in the presence of SDS and beta-ME. In at least two of the species examined, these appeared to be reaction center proteins. The conditions causing the proteins to become insoluble were complex and involved temperature, SDS concentration, and the presence of sulfhydryl reagents. The chromatophores of four of the Chromatiaceae species and two strains of one of the Rhodospirillaceae species examined had a protein-pigment complex that was visible in SDS-polyacrylamide gel profiles of samples dissolved at room temperature but was absent in samples dissolved at 100 degrees C.     

Sedimentation velocity to characterize surfactants and solubilized membrane proteins

Analytical ultracentrifugation sedimentation velocity, which combines the separation of the macromolecules and the analysis of their transportation to reach a rigorous thermodynamics study offers a robust tool for characterizing the homogeneity and association state of membrane protein. Samples of solubilized membrane proteins are indeed complex multi-component systems where detergent micelles and protein-detergent complexes coexist in solution, with associated lipids in variable amounts. We present here the sedimentation velocity theoretical background, the principle of the data analysis and the interpretation relevant for the study of membrane proteins. The results section presents examples and refers to published work. High resolution particle distribution are obtained from measurements in absorbance and interference, which permits the characterization of protein-detergent complexes-in terms of association state and bound detergents/lipids, even in heterogeneous samples, and of surfactants. We emphasize the precaution to be taken before the analysis, and the limits of the analysis. We show how the sedimentation velocity performed in H(2)O and D(2)O solvents may help to acertain the association state of solubilized membrane proteins. We discuss the complementarity with sedimentation equilibrium, density measurement, and size exclusion chromatography combined if necessary with the use of radiolabelled detergent or light scattering detection.     

Use of detergents in two-dimensional crystallization of membrane proteins

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.     

Topogenic signals in integral membrane proteins

Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic.proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information.     

Interaction of membrane proteins and lipids with solubilizing detergents

Detergents are indispensable in the isolation of integral membrane proteins from biological membranes to study their intrinsic structural and functional properties. Solubilization involves a number of intermediary states that can be studied by a variety of physicochemical and kinetic methods; it usually starts by destabilization of the lipid component of the membranes, a process that is accompanied by a transition of detergent binding by the membrane from a noncooperative to a cooperative interaction already below the critical micellar concentration (CMC). This leads to the formation of membrane fragments of proteins and lipids with detergent-shielded edges. In the final stage of solubilization membrane proteins are present as protomers, with the membrane inserted sectors covered by detergent. We consider in detail the nature of this interaction and conclude that in general binding as a monolayer ring, rather than as a micelle, is the most probable mechanism. This mode of interaction is supported by neutron diffraction investigations on the disposition of detergent in 3-D crystals of membrane proteins. Finally, we briefly discuss the use of techniques such as analytical ultracentrifugation, size exclusion chromatography, and mass spectrometry relevant for the structural investigation of detergent solubilized membrane proteins.     

Extraction of detergents from hydrophobic proteins with isopentanol: application to electrophoretic analysis of photosynthetic bacterial hydrophobic proteins

The method for extracting Triton X-100 used by I. H. Mather and C. B. Tampling [Anal. Biochem. 93, 139-142 (1979)], has been extended to other detergents of different charge and chemical nature. All the detergents tested can be extracted with isopentanol in conditions in which not more than 8% of hydrophobic or hydrophilic protein is lost from the water phase. The removal of detergent from reaction centers and light harvesting protein-pigment complexes of photosynthetic bacteria, eliminates the artifacts of oligomers when analyzed by sodium dodecyl sulfate-gel electrophoresis.     

Structuring detergents for extracting and stabilizing functional membrane proteins

Background

Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.

Methodology/Principal Findings

Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by ¹H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.

Conclusion/Significance

These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.     

FlgM proteins from different bacteria exhibit different structural characteristics

Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not require a stable structure for function. The importance of IDPs in many biological processes has been established but there remain unanswered questions about their evolution and conservation of their disordered state within a protein family. Our group has been studying the structural similarities among orthologous FlgM proteins, a model class of IDPs. We have previously shown that the FlgM protein from the thermophile Aquifex aeolicus has more structure at A. aeolicus' physiological temperature (85 °C) than is observed for the Salmonella typhimurium FlgM, suggesting that the disordered nature of FlgM varies among organisms and is not universally conserved. In this work, we extend these studies to the FlgM proteins from Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Bacillus subtilis. We demonstrate that the B. subtilis, E. coli, and S. typhimurium FlgMs exist in a premolten globule-like conformation, though the B. subtilis FlgM is in a more compacted conformation than the other two. The P. aeruginosa and P. mirabilis FlgM proteins exist in a currently unknown conformation that is not either coil-like or premolten globule-like. The P. aeruginosa FlgM appears to contain more weak intramolecular contacts given its more compacted state than the P. mirabilis FlgM. These results provide experimental evidence that members of the same protein family can exhibit different degrees of disorder, though understanding how different disordered states evolve in the same protein family will require more study.     

Immunoblotting of hydrophobic integral membrane proteins

For diagnosis and research purposes it is frequently desirable to measure by immunoblotting small amounts of proteins in complex mixtures such as tissue biopsy homogenates. Standard immunoblot procedures that give excellent results for soluble proteins unexpectedly gave low and irreproducible signals with some hydrophobic membrane proteins. We found that this was due to inefficient electrophoretic transfer to nitrocellulose, which could be corrected by modification of the transblot buffer. Hydrophobic integral membrane proteins of peroxisomes as well as other rat and human liver proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. The nitrocellulose-bound proteins were detected both by staining and by immunoblotting with an antiserum against the 22-kDa integral membrane protein of peroxisomes plus 125I-labeled protein A. A modified transblot buffer with 0.7 M glycine and 25 mM Tris (pH 7.7) but no methanol allowed use of a much shorter transfer time and strikingly improved the electrophoretic transfer of membrane proteins such that a peroxisomal integral membrane protein could be easily detected in human liver biopsy homogenates.