Membrane Proteins In Bilayers Research Articles

Advantages and potential limitations of applying AFM kymograph analysis to pharmaceutically relevant membrane proteins in lipid bilayers

Membrane proteins play critical roles in disease and in the disposition of many pharmaceuticals. A prime example is P-glycoprotein (Pgp) which moves a diverse range of drugs across membranes and out of the cell before a therapeutic payload can be delivered. Conventional structural biology methods have provided a valuable framework for comprehending the complex conformational changes underlying Pgp function, which also includes ATPase activity, but the lack of real-time information hinders understanding. Atomic force microscopy (AFM) is a single-molecule technique that is well-suited for studying active membrane proteins in bilayers and is poised to advance the field beyond static snapshots. After verifying Pgp activity in surface-support bilayers, we used kymograph analysis in conjunction with AFM imaging and simulations to study structural transitions at the 100 ms timescale. Though kymographs are frequently employed to boost temporal resolution, the limitations of the method have not been well characterized, especially for sparse non-crystalline distributions of pharmaceutically relevant membrane proteins like Pgp. Common experimental challenges are analyzed, including protein orientation, instrument noise, and drift. Surprisingly, a lateral drift of 75% of the protein dimension leads to only a 12% probability of erroneous state transition detection; average dwell time error achieves a maximum value of 6%. Rotational drift of proteins like Pgp, with azimuthally-dependent maximum heights, can lead to artifactual transitions. Torsional constraints can alleviate this potential pitfall. Confidence in detected transitions can be increased by adding conformation-altering ligands such as non-hydrolysable analogs. Overall, the data indicate that AFM kymographs are a viable method to access conformational dynamics for Pgp, but generalizations of the method should be made with caution.

Synthetic Biology-Based Solution NMR Studies on Membrane Proteins in Lipid Environments.

Although membrane proteins are in the focus of biochemical research for many decades the general knowledge of this important class is far behind soluble proteins. Despite several recent technical developments, the most challenging feature still is the generation of high-quality samples in environments suitable for the selected application. Reconstitution of membrane proteins into lipid bilayers will generate the most native-like environment and is therefore commonly desired. However, it poses tremendous problems to solution-state NMR analysis due to the dramatic increase in particle size resulting in high rotational correlation times. Nevertheless, a few promising strategies for the solution NMR analysis of membrane inserted proteins are emerging and will be discussed in this chapter. We focus on the generation of membrane protein samples in nanodisc membranes by cell-free systems and will describe the characteristic advantages of that platform in providing tailored protein expression and folding environments. We indicate frequent problems that have to be overcome in cell-free synthesis, nanodisc preparation, and customization for samples dedicated for solution-state NMR. Detailed instructions for sample preparation are given, and solution NMR approaches suitable for membrane proteins in bilayers are compiled. We further discuss the current strategies applied for signal detection from such difficult samples and describe the type of information that can be extracted from the various experiments. In summary, a comprehensive guideline for the analysis of membrane proteins in native-like membrane environments by solution-state NMR techniques will be provided.

Nanoscale dynamics of phospholipids reveals an optimal assembly mechanism of pore-forming proteins in bilayer membranes.

Cell membranes are believed to be highly complex dynamical systems having compositional heterogeneity involving several types of lipids and proteins as the major constituents. This dynamical and compositional heterogeneity is suggested to be critical to the maintenance of active functionality and response to chemical, mechanical, electrical and thermal stresses. However, delineating the various factors responsible for the spatio-temporal response of actual cell membranes to stresses can be quite challenging. In this work we show how biomimetic phospholipid bilayer membranes with variable lipid fluidity determine the optimal assembly mechanism of the pore-forming protein, listeriolysin O (LLO), belonging to the class of cholesterol dependent cytolysins (CDCs). By combining atomic force microscopy (AFM) and super-resolution stimulated emission depletion (STED) microscopy imaging on model membranes, we show that pores formed by LLO in supported lipid bilayers can have variable conformation and morphology depending on the fluidity of the bilayer. At a fixed cholesterol concentration, pores formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes were larger, flexible and more prone to coalescence when compared with the smaller and more compact pores formed in the lower fluidity 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. In contrast, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes did not show any evidence of pore formation. Fluorescence correlation spectroscopy (FCS) in STED mode revealed the appearance of a length scale, ξ, below which lipid dynamics, under the influence of LLO protein binding and assembly, becomes anomalous. Interestingly, the magnitude of ξ is found to correlate with both lipid fluidity and pore dimensions (and flexibility) in DOPC and POPC bilayers. However this length scale dependent crossover, signalling the onset of anomalous diffusion, was not observed in DMPC bilayers. Our study highlights the subtle interplay of lipid membrane mediated protein assembly and lipid fluidity in determining proteo-lipidic complexes formed in biomembranes and the significant insight that STED microscopy provides in unraveling critical aspects of nanoscale membrane biophysics.

Structure Determination of a Membrane Protein in Proteoliposomes

An NMR method for determining the three-dimensional structures of membrane proteins in proteoliposomes is demonstrated by determining the structure of MerFt, the 60-residue helix-loop-helix integral membrane core of the 81-residue mercury transporter MerF. The method merges elements of oriented sample (OS) solid-state NMR and magic angle spinning (MAS) solid-state NMR techniques to measure orientation restraints relative to a single external axis (the bilayer normal) from individual residues in a uniformly (13)C/(15)N labeled protein in unoriented liquid crystalline phospholipid bilayers. The method relies on the fast (>10(5) Hz) rotational diffusion of membrane proteins in bilayers to average the static chemical shift anisotropy and heteronuclear dipole-dipole coupling powder patterns to axially symmetric powder patterns with reduced frequency spans. The frequency associated with the parallel edge of such motionally averaged powder patterns is exactly the same as that measured from the single line resonance in the spectrum of a stationary sample that is macroscopically aligned parallel to the direction of the applied magnetic field. All data are collected on unoriented samples undergoing MAS. Averaging of the homonuclear (13)C/(13)C dipolar couplings, by MAS of the sample, enables the use of uniformly (13)C/(15)N labeled proteins, which provides enhanced sensitivity through direct (13)C detection as well as the use of multidimensional MAS solid-state NMR methods for resolving and assigning resonances. The unique feature of this method is the measurement of orientation restraints that enable the protein structure and orientation to be determined in unoriented proteoliposomes.

Thermodynamic Properties of Single Ion Channel Formation: Gramicidin

Single molecule fluorescence imaging has been used to unequivocally differentiate between rhodamine-6G labeled gramicidin monomeric subunits and channel-forming dimers. Absolute identification of individual particles was achieved by accounting for both particle diffusion and intensity, with dimer intensity being twice that of the monomers. In accordance with current diffusion models of proteins in bilayer membranes, we observed dimers to diffuse more slowly through the bilayer than the monomers and have reported diffusion coefficients of 1.2 × 10-8 and 3.5 × 10-8 cm2/s for the dimers and monomers, respectively. By correlating the diffusion data with measured fluorescence intensities of the tracked particles, it was possible to determine the distribution of monomers and dimers within the bilayer at various temperatures. The results allow complete characterization of the thermodynamic properties of dimer formation, 2G1 ↔ G2, necessary for channel function. Reported are the temperature-dependent equilibrium ...

NMR-Based Amide Hydrogen–Deuterium Exchange Measurements for Complex Membrane Proteins: Development and Critical Evaluation

A method for measuring site-specific amide hydrogen–deuterium exchange rates for membrane proteins in bilayers is reported and evaluated. This method represents an adaptation and extension of the approach of Dempsey and co-workers (Biophys. J. 70, 1777–1788 (1996)) and is based on reconstituting 15N-labeled membrane proteins into phospholipid bilayers, followed by lyophilization and rehydration with D2O or H2O (control). Following incubation for a time t under hydrated conditions, samples are again lyophilized and then solubilized in an organic solvent system, where 1H–15N HSQC spectra are recorded. Comparison of spectra from D2O-exposed samples to spectra from control samples yields the extent of the H–D exchange which occurred in the bilayers during time t. Measurements are site specific if specific 15N labeling is used. The first part of this paper deals with the search for a suitable solvent system in which to solubilize complex membrane proteins in an amide “exchange-trapped” form for NMR quantitation of amide peak intensities. The second portion of the paper documents application of the overall procedure to measuring site-specific amide exchange rates in diacylglycerol kinase, a representative integral membrane protein. Both the potential usefulness and the significant limitations of the new method are documented.

Effect of cholesterol, fatty acyl chain composition, and bilayer curvature on the interaction of cytochrome b5 with liposomes of phosphatidylcholines.

To determine the effect of cholesterol and lipid packing on the solubility of membrane proteins in bilayers, cytochrome b5 incorporation into phosphatidylcholine (PC) liposomes was determined as a function of bilayer curvature (SUVs versus LUVs), fatty acyl chain composition, and cholesterol content. The equilibrium affinity constant for the formation of a 1:1 b5/PC complex, Kp, and the number of PC's per "site" at saturation, n, were determined from binding isotherms, which were obtained by measuring the increase in intrinsic tryptophan fluorescence. With LUVs, n was also determined directly by gel filtration. The following results were obtained: (1) Both Kp and the saturating level of b5 binding, s (n-1), are significantly greater for SUVs than for LUVs. In LUVs, a binding site must consist of several surrounding lipid layers. (2) Cholesterol reduces Kp and s by factors that range from 1 to > 100. Binding inhibition is highly sensitive to the liposome size and to the fatty acyl composition of the PC; the latter correlates with the condensing effects on PC: C1satC2mono > C1satC2di approximately natural mixtures > C1unsat-C2unsat. (3) With POPC LUVs, the binding inhibition was 3.6-, 1.4- and 17-fold within the ranges of 0-20, 20-33, and 33-50 mole percent cholesterol, respectively. (4) The equilibrium binding constant to SUVs is greater for liposomes that are prepared from natural PC mixtures than for vesicles of a single synthetic phospholipid. The reductions in b5 binding correlate with reductions in bilayer free volume, which were calculated from monolayer studies of the lipid mixtures. The sensitivity of liposome saturability to bilayer curvature, fatty acyl chain composition, and cholesterol content may account for the disparate results among previous studies of cholesterol-protein interactions. A more significant implication is that in biological membranes with high levels of cholesterol, subtle variations in the fatty acyl chain composition could substantially affect the solubility and physical states of integral membrane proteins.

Biochemical properties of a 24 kilodalton membrane glycoprotein antigen complex from Schistosoma mansoni

Antibodies were affinity purified from crude antiserum by elution from the 24 kDa region of preparative one-dimensional Western blots containing immobilized adult Schistosoma mansoni inner bilayer membrane proteins. They were shown to be specific for a single acidic polypeptide complex, Smgp24, following immunoblotting from two-dimensional polyacrylamide gels. These antibodies were then used to detect the presence of the Smgp24 complex in fractions prepared from lectin affinity chromatography, phase separation in Triton X-114 and chemical and enzymatic carbohydrate modification treatments. The 24 kDa antigen was bound and specifically eluted from both concanavalin A and lentil lectin affinity matrices. In addition, the electrophoretic mobility of the antigen was shifted to approximately 20 kDa after treatment with endoglycosidase F and N-glycanase, but was not appreciably altered following treatment with endoglycosidase H, neuraminidase, or sodium meta-periodate. The 20 kDa species produced by endoglycosidase F or N-glycanase treatment no longer bound to the lectin affinity resins. The Smgp24 complex also partitioned almost quantitatively into the detergent-enriched phase after phase separation in Triton X-114 solutions. These results indicate that the Smgp24 complex is an antigenic integral membrane glycoprotein and may consist of a single polypeptide backbone which is extensively post- or co-translationally modified.

Lateral diffusion of lipids and proteins in bilayer membranes

Current knowledge of lipid and protein diffusion rates in homogeneous, fluid, artificial bilayers is reviewed and the ability of current theory to account for these rates is evaluated as a reference for understanding lateral diffusion in biomembranes. Experimental evidence on the effects of increasing protein concentration on the lateral diffusion of membrane lipids and proteins is summarized to provide a qualitative description of the situation in biomembranes. Lipid bilayers Lateral diffusion Membrane proteins Fluorescent probes Photobleaching