Membrane Proteins In Micelles Research Articles

The Thermodynamic Stability of Membrane Proteins in Micelles and Lipid Bilayers Investigated with the Ferrichrom Receptor FhuA

Extraction of integral membrane proteins into detergents for structural and functional studies often leads to a strong loss in protein stability. The impact of the lipid bilayer on the thermodynamic stability of an integral membrane protein in comparison to its solubilized form in detergent was examined and compared for FhuA from Escherichia coli and for a mutant, FhuAΔ5-160, lacking the N-terminal cork domain. Urea-induced unfolding was monitored by fluorescence spectroscopy to determine the effective free energies Delta G{^text{o}_{rm u}} of unfolding. To obtain enthalpic and entropic contributions of unfolding of FhuA, Delta G{^text{o}_{rm u}} were determined at various temperatures. When solubilized in LDAO detergent, wt-FhuA and FhuAΔ5-160 unfolded in a single step. The 155-residue cork domain stabilized wt-FhuA by DeltaDelta G{^text{o}_{rm u}} ~ 40 kJ/mol. Reconstituted into lipid bilayers, wt-FhuA unfolded in two steps, while FhuAΔ5-160 unfolded in a single step, indicating an uncoupled unfolding of the cork domain. For FhuAΔ5-160 at 35 °C, Delta G{^text{o}_{rm u}} increased from ~ 5 kJ/mol in LDAO micelles to about ~ 20 kJ/mol in lipid bilayers, while the temperature of unfolding increased from TM ~ 49 °C in LDAO micelles to TM ~ 75 °C in lipid bilayers. Enthalpies Delta H{_{rm M}^text{o}}were much larger than free energies Delta G{^text{o}_{rm u}} , for FhuAΔ5-160 and for wt-FhuA, and compensated by a large gain of entropy upon unfolding. The gain in conformational entropy is expected to be similar for unfolding of FhuA from micelles or bilayers. The strongly increased TM and Delta H{_{rm M}^text{o}} observed for the lipid bilayer-reconstituted FhuA in comparison to the LDAO-solubilized forms, therefore, very likely arise from a much-increased solvation entropy of FhuA in bilayers.Graphical abstract

Exploring the Molecular Machinery of Denitrification in Haloferax mediterranei Through Proteomics.

Many proteins and enzymes involved in denitrification in haloarchaea can be inferred to be located between the cytoplasmic membrane and the S-layer, based on the presence of a Tat signal sequence and the orientation of the active site that some of these enzymes have. The membrane fraction of the haloarchaeon Haloferax mediterranei (R-4), grown under anaerobic conditions in the presence of nitrate, was solubilized to identify the respiratory proteins associated or anchored to it. Using Triton X-100, CHAPS, and n-Octyl-β-d-glucopyranoside at different concentrations we found the best conditions for isolating membrane proteins in micelles, in which enzymatic activity and stability were maintained. Then, they were subjected to purification using two chromatographic steps followed by the analysis of the eluents by NANO-ESI Chip-HPLC-MS/MS. The results showed that the four main enzymes of denitrification (nitrate, nitrite, nitric oxide, and nitrous oxide reductases) in H. mediterranei were identified and they were co-purified thanks to the micelles made with Triton X-100 (20% w/v for membrane solubilisation and 0.2% w/v in the buffers used during purification). In addition, several accessory proteins involved in electron transfer processes during anaerobic respiration as well as proteins supporting ATP synthesis, redox balancing and oxygen sensing were detected. This is the first characterization of anaerobic membrane proteome of haloarchaea under denitrifying conditions using liquid chromatography-mass spectrometry. It provides new information for a better understanding of the anaerobic respiration in haloarchaea.

Measuring Remodeling of the Lipid Environment Surrounding Membrane Proteins with Lipid Exchange and Native Mass Spectrometry.

Due to their crucial biochemical roles, membrane proteins are important drug targets. Although it is clear that lipids can influence membrane protein function, the chemistry of lipid binding remains difficult to study because protein-lipid interactions are polydisperse, competitive, and transient. Furthermore, detergents, which are often used to solubilize membrane proteins in micelles, may disrupt lipid interactions that occur in bilayers. Here, we present two new approaches to quantify protein-lipid interactions in bilayers and understand how membrane proteins remodel their surrounding lipid environment. First, we used mass spectrometry (MS) to measure the exchange of lipids between lipoprotein nanodiscs with and without an embedded membrane protein. Shifts in the lipid distribution toward the membrane protein nanodiscs revealed lipid binding, and titrations allowed measurement of the optimal lipid composition for the membrane protein. Second, we used native or nondenaturing MS to ionize membrane protein nanodiscs with heterogeneous lipids. Ejecting the membrane protein complex with bound lipids in the mass spectrometer revealed enrichment of specific lipids around the membrane protein. Both new approaches showed that the E. coli ammonium transporter AmtB prefers phosphatidylglycerol lipids overall but has a minor affinity for phosphatidylcholine lipids.

Quantification of Membrane Protein Self-Association with a High-Throughput Compatible Fluorescence Assay.

For many membrane proteins, self-association serves both structural and functional roles. Studies of such association can be simplified by switching to micelles as the membrane-mimicking environment, but native interaction is not preserved in all detergents. The selection of suitable conditions for biochemical experiments would be greatly facilitated by a quantitative high-throughput assay. Here we showed that the fluorescence polarization reduction, which resulted from homo-Förster resonance energy transfer and was measured in a high-throughput compatible format, can be used to determine both association states and constants for membrane proteins in micelles.

EPR Spectroscopic Distance Measurements of the KCNE1 Membrane Protein in Micelles and Lipid Bilayers

Distance measurements between two strategically placed nitroxide spin labels can provide pertinent structural information on complicated biological systems such as membrane proteins. Distances of approximately 8 A - 25 A can be determined by measuring line broadening due to dipolar coupling using continuous wave (CW)-EPR spectroscopy. The short range distances can be measured between isotopically coupled nuclear spins and nitroxide electronic spin labels up to a distance of about 8 A using Electron Spin Echo Envelope Modulation (ESEEM) and long range distances of 20 A - 70A between two nitroxide electronic spin labels using Double Electron Electron Resonance (DEER). The transmembrane domain (TMD) of KCNE1 binds to the pore domain of the KCNQ1 channel modulating the channel's gating. In order to characterize the structural properties of the TMD of KCNE1, double cysteine mutants were prepared for TMD of KCNE1 and further modified by the addition of a MTSL nitroxide spin label. The purified proteins were reconstituted into LMPG detergent micelles and POPC/POPG bilayer vesicles. Dipolar broadening CW-EPR experiments were performed on the double mutants F53C-L63C, F56C-S64C, L59C-Y65C, and L59C-S64C at 225K; and DEER experiments on double mutants V50C-S68C, V47C-I66C at 80K. Molecular Dynamics Modeling study was performed on these mutants using nanoscale molecular dynamics (NAMD) with the molecular graphics software VMD. A 1 ns molecular dynamics simulation was ran for each double mutants and distance distribution was obtained by analyzing the trajectory data file using VMD. The distance distributions obtained from dipolar broadening CW EPR and DEER are in good agreement with NAMD/VMD and MMM modeling results.

Structural similarity of a membrane protein in micelles and membranes.

The anisotropic spin interactions measured for membrane proteins in weakly oriented micelles and in oriented lipid bilayers provide independent and potentially complementary high-resolution restraints for structure determination. Here we show that the membrane protein CHIF adopts a similar structure in lipid micelles and bilayers, allowing the restraints from micelle and bilayer samples to be combined in a complementary fashion to enhance the structural information. Back-calculation and assignment of the NMR spectrum of CHIF in oriented lipid bilayers, from the structure determined in micelles, provides additional restraints for structure determination as well as the global orientation of the protein in the membrane. The combined use of solution and solid-state NMR restraints also affords cross-validation for the structural analysis.

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.

The GxxxG-Containing Transmembrane Domain of the CCK4 Oncogene Does Not Encode Preferential Self-Interactions

The recently cloned colon carcinoma kinase 4 (CCK4) oncogene contains an evolutionarily conserved GxxxG motif in its single transmembrane domain (TMD). It has previously been suggested that this pairwise glycine motif may provide a strong driving force for transmembrane helix-helix interactions. Since CCK4 is thought to represent a new member of the receptor tyrosine kinase family, interactions between the TMDs may be important in receptor self-association and activation of signal transduction pathways. To determine whether this conserved CCK4 TMD can drive protein-protein interactions, we have carried out a thermodynamic study using the TMD expressed as a Staphylococcal nuclease (SN) fusion protein. Similar SN-TMD fusion proteins have been used to determine the sequence specificity and thermodynamics of transmembrane helix-helix interactions in a number of membrane proteins, including glycophorin A. Using sedimentation equilibrium in C14 betaine micelles, we discovered that the CCK4 TMD is unable to drive strong protein-protein interactions. At high protein/detergent ratios, the SN-CCK4 fusion protein will dimerize, but a stochastic model for protein association in micelles can explain the observed dimer population. For low-affinity interactions such as the one studied here, an understanding of this discrete stochastic distribution of membrane proteins in micelles is important for distinguishing between preferential and random self-interactions, which can both influence the oligomeric population. The lack of a thermodynamically meaningful self-association propensity for the CCK4 TMDs demonstrates that a GxxxG motif is not sufficient to drive transmembrane helix-helix interactions.

Weak alignment of membrane proteins in stressed polyacrylamide gels

Residual dipolar couplings are important as angular constraints for the structure determination of membrane proteins in micelles. Strained polyacrylamide gels are one of the few available mechanisms available for inducing the requisite weak alignment for these samples. However, their use is frequently limited by the ability to incorporate proteins and buffer solutions into the gel matrix. The implementation of several methods of incorporating membrane proteins into gels are described. Conditions for copolymerizing the protein in the absence of a change in pH are detailed. Electrophoresis is also shown to be a useful method to incorporate proteins. Weak alignment of the protein–micelle complex in the gel matrix is subsequently achieved using either vertical or radial compression. The magnitude of alignment can be controlled by altering the gel concentration, the acrylamide/bisacrylamide ratio, and the compression ratio. The alignment tensor can be altered relative to uncharged polyacrylamide gels by copolymerizing samples with acrylamide/acrylic acid to incorporate negative charges in the strained polyacrylamide gel to provide an alternate orientation

14] Nuclear magnetic resonance of membrane-associated peptides and proteins

Structural biology is based on the premise that the fundamental understanding of biological functions lies in the three-dimensional structures of proteins and other biopolymers. The two well-established experimental methods for determining the structures of proteins work very well for globular proteins: witness the explosive growth of the Protein Data Bank (PDB). However, approximately 30% of all expressed polypeptides are membrane-associated, and neither X-ray crystallography nor solution nuclear magnetic resonance (NMR) spectroscopy is very effective for these proteins. The lipids required for the structural integrity and functionality of membrane proteins impede crystallization as well as the rate of overall reorientation in solution. NMR of Proteins NMR spectroscopy can be applied to wide variety of samples, ranging from isotropic solutions to crystalline powders, including those with slowly reorienting or immobile macromolecules, such as membrane proteins in lipid environments. NMR is capable of resolving signals from all atomic sites in proteins, and each site has several well-characterized nuclear spin interactions that can be used as sources of information about molecular structure and dynamics, as well as chemical interactions. The spin interactions can be probed through radio frequency (rf) irradiations and sample manipulations that lead to complementary strategies for NMR spectroscopy of membrane proteins reconstituted in lipid micelles or bilayers. Comparisons between the results obtained with solution NMR experiments on lipid micelle samples, and solid-state NMR experiments on lipid bilayer samples, are especially valuable for membrane proteins with predominantly helical secondary structure. Multidimensional solution NMR methods can be successfully applied to relatively small membrane proteins in micelles; however, the size limitation is substantially more severe than for globular proteins because the many lipid molecules associated with each polypeptide slow its overall reorientation rate. In particular, using currently available instruments and methods, it is difficult to resolve, assign, and measure the “long-range” nuclear overhauser effects (NOEs) between hydrogens on hydrophobic side-chains that are needed to determine tertiary structures based on distance constraints. However, the ability to weakly align membrane proteins in micelles enables the measurement of residual dipolar couplings, and improves the feasibility of determining the structures of membrane proteins using solution NMR methods. Nonetheless, it is highly desirable to determine the structures of membrane proteins in the definitive environment of phospholipid bilayers, where solution NMR methods fail completely for all classes of membrane proteins. Fortunately, solid-state NMR spectroscopy is well suited for peptides and proteins immobilized in phospholipid bilayers. Both oriented sample and magic angle spinning methods provide approaches to measuring orientational and distance parameters for structure determination.

Lanthanide Ion Binding to Adventitious Sites Aligns Membrane Proteins in Micelles for Solution NMR Spectroscopy

Lanthanide Ion Binding to Adventitious Sites Aligns Membrane Proteins in Micelles for Solution NMR Spectroscopy

Lanthanide ions bind specifically to an added "EF-hand" and orient a membrane protein in micelles for solution NMR spectroscopy.

Twelve amino acid residues corresponding to an "EF-hand" calcium-binding site were added to the N-terminus of a protein, providing a specific lanthanide ion binding that weakly orients the protein in solution. A comparison of spectra of the protein with and without the EF-hand residues demonstrates that the structure of the native protein is not perturbed by this modification, since there are minimal chemical shift changes. With a lanthanide but not calcium bound to the EF-hand, the protein is weakly oriented by the magnetic field, since residual dipolar couplings can be measured. Since the signs and magnitudes of the couplings varied with the type of lanthanide, this demonstrated the ability to obtain multiple orientations of the protein in solution. The sample is a membrane protein in lipid micelles that disrupted the commonly employed bicelle and filamentous phage solutions; therefore, the addition of a specific metal binding site in the form of an EF-hand may provide a general approach to orienting proteins where the addition of external agents is problematic. An additional benefit is that the lanthanide ions perturb the protein resonances in ways that provide unique orientational and distance constraints.

NMR structural studies of membrane proteins

The three-dimensional structures of membrane proteins are essential for understanding their functions, interactions and architectures. Their requirement for lipids has hampered structure determination by conventional approaches. With optimized samples, it is possible to apply solution NMR methods to small membrane proteins in micelles; however, lipid bilayers are the definitive environment for membrane proteins and this requires solid-state NMR methods. Newly developed solid-state NMR experiments enable completely resolved spectra to be obtained from uniformly isotopically labeled membrane proteins in phospholipid lipid bilayers. The resulting operational constraints can be used for the determination of the structures of membrane proteins.

Escherichia coli diacylglycerol kinase: a case study in the application of solution NMR methods to an integral membrane protein

Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.