Misfolding of membrane proteins in health and disease: the lady or the tiger?

Protein misfolding is increasingly recognized as a factor in many diseases, including cystic fibrosis, Parkinson's, Alzheimer's and atherosclerosis. Many proteins involved in misfolding-based pathologies are membrane-associated, such that the bilayer may play roles in normal and aberrant folding. It can be argued that the in vivo partitioning of eukaryotic membrane proteins between folding and misfolding pathways is under kinetic control. Moreover, the balance between these pathways can be surprisingly delicate.

Customizing model membranes and samples for NMR spectroscopic studies of complex membrane proteins.

Both solution and solid state nuclear magnetic resonance (NMR) techniques for structural determination are advancing rapidly such that it is possible to contemplate bringing these techniques to bear upon integral membrane proteins having multiple transmembrane segments. This review outlines existing and emerging options for model membrane media for use in such studies and surveys the special considerations which must be taken into account when preparing larger membrane proteins for NMR spectroscopic studies.

Thiol modification of diacylglycerol kinase: dependence upon site membrane disposition and reagent hydrophobicity.

Reaction rates were determined between disulfide reagents of varying hydrophobicity and single-cysteine mutants of diacylglycerol kinase, an integral membrane protein. Polar reagents reacted most rapidly with surface-exposed sites. However, a very non-polar reagent also reacted more rapidly with exposed cysteines than with membrane sites. Moreover, this non-polar reagent usually reacted more slowly with membrane sites than did more polar reagents. These results are consistent with the notion that disulfide exchange reactions involving buried cysteines of diacylglycerol kinase are very slow in the membrane interior, such that the competing rates of reactions which occur when normally buried cysteine sites make motional excursions to hydrated regions of the interface can be significant.

Use of amphipathic polymers to deliver a membrane protein to lipid bilayers.

Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.

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 (15)N-labeled membrane proteins into phospholipid bilayers, followed by lyophilization and rehydration with D(2)O or H(2)O (control). Following incubation for a time t under hydrated conditions, samples are again lyophilized and then solubilized in an organic solvent system, where (1)H-(15)N HSQC spectra are recorded. Comparison of spectra from D(2)O-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 (15)N 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.

Qualitative comparison of the bilayer-associated structures of diacylglycerol and a fluorinated analog based upon oriented sample NMR data.

sn-1,2-Dimyristoylglycerol (DMDAG) and sn-1,2-dimyristoyl-3-fluoropropanediol (DMFPD) were synthesized in carbonyl 13C-labeled and acyl chain perdeuterated forms. These compounds were reconstituted at low levels into both randomly dispersed dimyristoylphosphatidylcholine (DMPC) bilayers and magnetically orientable DMPC media. Samples were subjected to NMR analysis, leading to a substantial body of 2H quadrupolar splitting, 13C-13C and 13C-19F dipolar coupling and 13C chemical shift anisotropy data for both compounds. A number of measurements were also made for DMPC. The data acquired from magnetically oriented samples were found to be undesirably affected by the presence of artifacts related to the experimental use of the oriented lipid media. However, it was possible to draw a number of qualitative conclusions from the data and to correct the data so that they may ultimately prove useful in quantitative structural analyses of bilayer-associated DMDAG and DMFPD. Comparison of the DMDAG data with corresponding measurements of DMPC suggests a high degree of similarity between the two compounds within bilayers composed primarily of L alpha phase phosphatidylcholine, consistent with previous work (S.O. Smith et al. (1992) Biochemistry 31, 11660-11664). Comparison of the data for DMDAG and DMFPD suggests that the hydroxyl proton of DMDAG is involved in hydrogen bonding interactions, which appear to be largely responsible for maintaining the orientational inequivalence of its two acyl chains. Bilayer-associated DMFPD also appears to exhibit a higher degree of whole-molecule disorder than DMDAG, again suggestive of an important structural role for the hydroxyl proton of DMDAG. Finally, comparison of 13C-NMR data for oriented DMPC in the presence and absence of low levels of DMDAG and DMFPD indicated that neither compound induces a significant change in the averaged conformational state of DMPC comprising the bilayer matrix of the oriented samples.

Synthesis and characterization of a 13C-labeled alpha-mannosyl glycolipid analog from [13C]glucose.

A method is described for the synthesis of a [13C]alpha-mannosyl glycolipid analog from [13C]glucose. After acetylation and reduction of glucose (U-13C6, 30%) to tri-O-acetyl-D-glucal (U-13C6, 30%), addition of the nucleophile 2-[2-[2-[2-(tetradecyloxy)ethoxy]ethoxy]ethoxy]- ethanol (tetra-decyltetraglycol) yields the rearrangement product alpha-tetradecyltetraglycol 2,3-dideoxyl-4,6-di-O-acetyl-D-gluco- pyranoside (U-13C6, 30%). The rearrangement product is oxidized with osmium tetroxide to produce tetradecyltetraglycol alpha-mannoside (U-13C6, 30%). The interaction of the glycolipid with the plant lectin concanavalin A is characterized by a vesicle agglutination assay.

Substrate specificity of gamma-secretase and other intramembrane proteases.

Gamma-Secretase is a promiscuous protease that cleaves bitopic membrane proteins within the lipid bilayer. Elucidating both the mechanistic basis of gamma-secretase proteolysis and the precise factors regulating substrate identification is important because modulation of this biochemical degradative process can have important consequences in a physiological and pathophysiological context. Here, we briefly review such information for all major classes of intramembranously cleaving proteases (I-CLiPs), with an emphasis on gamma-secretase, an I-CLiP closely linked to the etiology of Alzheimer's disease. A large body of emerging data allows us to survey the substrates of gamma-secretase to ascertain the conformational features that predispose a peptide to cleavage by this enigmatic protease. Because substrate specificity in vivo is closely linked to the relative subcellular compartmentalization of gamma-secretase and its substrates, we also survey the voluminous body of literature concerning the traffic of gamma-secretase and its most prominent substrate, the amyloid precursor protein.

Solid state 13C NMR of unlabeled phosphatidylcholine bilayers: spectral assignments and measurement of carbon-phosphorus dipolar couplings and 13C chemical shift anisotropies.

The direct measurement of 13C chemical shift anisotropies (CSA) and 31P-13C dipolar splitting in random dispersions of unlabeled L alpha-phase phosphatidylcholine (PC) has traditionally been difficult because of extreme spectral boradening due to anisotropy. In this study, mixtures of dimyristoyl phosphatidylcholine (DMPC) with three different detergents known to promote the magnetic orientation of DMPC were employed to eliminate the powder-pattern nature of signals without totally averaging out spectral anisotropy. The detergents utilized were CHAPSO, Triton X-100, and dihexanoylphosphatidylcholine (DHPC). Using such mixtures, many of the individual 13C resonances from DMPC were resolved and a number of 13C-31P dipolar couplings were evident. In addition, differing line widths were observed for the components of some dipolar doublets, suggestive of dipolar/chemical shift anisotropy (CSA) relaxation interference effects. Oriented sample resonance assignments were made by varying the CHAPSO or DHPC to DMPC ratio to systematically scale overall bilayer order towards the isotropic limit. In this manner, peaks could be identified based upon extrapolation to their isotropic positions, for which assignments have previously been made (Lee, C.W.B., and R.G. Griffin. 1989. Biophys. J. 55:355-358; Forbes, J., J. Bowers, X. Shan, L. Moran, E. Oldfield, and M.A. Moscarello. 1988. J. Chem. Soc., Faraday, Trans. 1 84:3821-3849). It was observed that the plots of CSA or dipolar coupling versus overall bilayer order obtained from DHPC and CHAPSO titrations were linear. Estimates of the intrinsic dipolar couplings and chemical shift anisotropies for pure DMPC bilayers were made by extrapolating shifts and couplings from the detergent titrations to zero detergent. Both detergent titrations led to similar "intrinsic" CSAs and dipolar couplings. Results extracted from an oriented Triton-DMPC mixture also led to similar estimates for the detergent-free DMPC shifts and couplings. The results from these experiments were found to compare favorably with limited measurements made from pure L alpha PC. This detergent-based method for assigning spectra and for determining dipolar couplings and CSA in detergent-free systems should be extendable to other lipid systems. The resulting data set from this study may prove useful in future modeling of the structure and dynamics of DMPC bilayers. In addition, the fact that experiments utilizing each of the three detergents led to similar estimates for the spectral parameters of pure DMPC, and the fact that spectral parameter versus bilayer order plots were linear, indicate that the averaged conformation and dynamics of DMPC in the presence of the three detergents are very similar to those of pure L alpha DMPC.

Magnetically orientable phospholipid bilayers containing small amounts of a bile salt analogue, CHAPSO.

Buffered mixtures of the detergent 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) and dimyristoylphosphatidylcholine (DMPC) orient in the presence of a strong magnetic field over a wide range of water contents (at least 65-85%) and CHAPSO:DMPC molar ratios (typically 1:10-1:3). 31P NMR studies show that the phospholipid in such mixtures is oriented with its director axis perpendicular to the magnetic field. 31P and 2H NMR results also suggest that the structure and dynamics of the DMPC molecules are similar to that of pure phospholipids existing in the liquid crystalline (L alpha) bilayer phase. The ability of 1:5 CHAPSO:DMPC samples to orient is highly tolerant of large changes in temperature, pH, and ionic strength, as well as to the addition of substantial amounts of charged amphiphiles or soluble protein. However, 2H NMR studies of deuterated beta-dodecyl melibiose (DD-MB) solubilized in the system indicate the head group conformation and/or dynamics of this glycolipid analogue is dependent upon the CHAPSO concentration. Despite the latter results, the orientational versatility of the system, together with the nondenaturing properties of CHAPSO, makes this system useful in spectroscopic studies of membrane-associated phenomena.

Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies.

This paper describes a study undertaken to assess the possibility and practical consequences of reconstituting integral and peripheral membrane proteins into bilayered discoidal mixed micelles ("bicelles") composed of dimyristoylphosphatidylcholine and smaller amounts of either CHAPSO or short-chain phosphatidylcholine. The amphiphilic assemblies in these mixtures are uniquely suited for use in NMR structural studies because they can be magnetically oriented with experimentally-tunable system order. The first step of this study was to test about 15 membrane-associating polypeptides and proteins for their ability to interfere with magnetic orientation of the bicellar assemblies. A variety of results were obtained ranging from no perturbation to a complete disruption of orientation. Second, the suitability of bicelles as mimics of natural bilayers was tested by reconstituting diacylglycerol kinase, an integral membrane enzyme. The kinase was observed to be functional and completely stable for at least 24 h when incubated at 38 degrees C in bicelles. Third, the NMR spectra from a number of bicelle-reconstituted proteins were examined. In some cases, 13C NMR resonances from reconstituted proteins were extremely broad and asymmetric. In other cases, resonances from reconstituted proteins were moderately broad, but much less so than resonances from proteins reconstituted into multilayers oriented by mechanical methods. In the cases of two surface-associating proteins (cytochrome c and leucine enkephalin), oriented sample 13C NMR spectra of extremely high resolution were obtained. For these proteins it was also demonstrated that the experimentally variable order of the bicellar assemblies could be exploited to provide a means of screening for detergent-specific structural perturbations, for making spectral assignments, and for measuring chemical shift anisotropies and dipolar couplings. Taken as a whole, these results indicate that bicelles may be uniquely and effectively employed as model membranes to facilitate NMR structural studies of many, but not all, membrane proteins.

An approximate model and empirical energy function for solute interactions with a water-phosphatidylcholine interface.

An empirical model of a liquid crystalline (L alpha phase) phosphatidylcholine (PC) bilayer interface is presented along with a function which calculates the position-dependent energy of associated solutes. The model approximates the interface as a gradual two-step transition, the first step being from an aqueous phase to a phase of reduced polarity, but which maintains a high enough concentration of water and/or polar head group moieties to satisfy the hydrogen bond-forming potential of the solute. The second transition is from the hydrogen bonding/low polarity region to an effectively anhydrous hydrocarbon phase. The "interfacial energies" of solutes within this variable medium are calculated based upon atomic positions and atomic parameters describing general polarity and hydrogen bond donor/acceptor propensities. This function was tested for its ability to reproduce experimental water-solvent partitioning energies and water-bilayer partitioning data. In both cases, the experimental data was reproduced fairly well. Energy minimizations carried out on beta-hexyl glucopyranoside led to identification of a global minimum for the interface-associated glycolipid which exhibited glycosidic torsion angles in agreement with prior results (Hare, B.J., K.P. Howard, and J.H. Prestegard. 1993. Biophys. J. 64:392-398). Molecular dynamics simulations carried out upon this same molecule within the simulated interface led to results which were consistent with a number of experimentally based conclusions from previous work, but failed to quantitatively reproduce an available NMR quadrupolar/dipolar coupling data set (Sanders, C.R., and J.H. Prestegard. 1991. J. Am. Chem. Soc. 113:1987-1996). The proposed model and functions are readily incorporated into computational energy modeling algorithms and may prove useful in future studies of membrane-associated molecules.

Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR.

Mixtures of long-chain and short-chain phosphatidylcholine (PC) were characterized by multinuclear (13C, 31P, 2H) solid-state nuclear magnetic resonance. This work complements and extends previous characterization of such mixtures by focusing on concentrated mixtures at temperatures above the gel to liquid crystalline phase transition temperature (Tm) of the long-chain PC component. Above Tm it was observed that highly oriented, bilayer-like assemblies could be formed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in molar ratios ranging from approximately 1:3.5 to 1:2 (DHPC:DMPC) over a considerable range of lipid concentrations (at least 3-40% w/v total lipid, for a 1:2.5 sample). Orientation was observed to occur only in an L alpha-like phase. The NMR data can be accounted for by a general model of the DHPC-DMPC aggregates in which DHPC can be found in two distinct populations (one highly ordered, one not). The averaged conformations of the glycerol backbone/headgroup regions of the long- and short-chain PC composing the assemblies were judged by solid-state 13C NMR to be similar to each other. The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.

Escherichia coli diacylglycerol kinase is an evolutionarily optimized membrane enzyme and catalyzes direct phosphoryl transfer.

In this contribution the kinetic mechanism and substrate specificity of Escherichia coli diacylglycerol kinase were examined. Steady state kinetic studies were carried out under mixed micellar conditions using a novel continuous coupled assay system. The kinetic data were consistent with a random equilibrium mechanism, implying that diacylglycerol kinase catalyzes direct phosphoryl transfer from MgATP to diacylglycerol. This was supported by failure to detect an enzyme-phosphate covalent intermediate and by the observation that the bisubstrate analog adenosine 5'-tetraphosphoryl-3-O-(1,2-dihexanoyl)-sn-glycerol inhibits the enzyme (Ki << Km,DAG). While diacylglycerol kinase's kcat/Km is modest compared with the efficiency of many water-soluble enzymes, the enzyme nevertheless appears to be an evolutionarily optimized biocatalyst in the sense that its chemical reaction rate approaches the substrate diffusion-controlled limit. The in vivo rate-limiting step of DAGK's reaction appears to be, in part, the transbilayer diffusion of diacylglycerol from the outer leaflet to the inner leaflet of the cytoplasmic membrane where DAGK's active site is located. DAGK was observed to maintain a high nucleotide substrate specificity, with most of this specificity being expressed in the form of reductions in kcat for ATP analogs.

Functionality of a membrane protein in bicelles.

Bicelles are bilayered discoidal lipid-detergent assemblies which are useful as model membranes. To date, there has been no direct demonstration of functional viability for an integral membrane protein reconstituted into bicelles. In this contribution, the catalytic activity of diacylglycerol kinase (DAGK) was measured following reconstitution into several different bicelle systems and compared to activities measured in traditional mixed micelles and vesicles. For the most optimal bicelle systems tested, DAGK activities approached those observed in mixed micelles or vesicles. For some other bicellar mixtures tested, activities were much lower, with steady-state kinetic data indicating reduced V(max) rather than perturbations in substrate K(m). Catalytically, DAGK showed a strong preference for bicelles containing 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) as the detergentcomponent relative to short-chained phosphatidylcholine.DAGK also exhibited a preference for dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine bicelles relative to those of dilauroylphosphatidylcholine.

Simulation of NMR data from oriented membrane proteins: practical information for experimental design.

Several hundred solid state NMR dipolar couplings and chemical shift anisotropies were simulated for the polytopic membrane protein, bacteriorhodopsin, and for an idealized transmembrane peptide conforming to several different secondary structures (alpha- and 3(10)-helices and parallel and antiparallel beta-sheets), each at several tilt angles with respect to the bilayer normal. The use of macroscopically oriented samples was assumed. The results of these simulations suggest: (i) Because of the r-3 dependence of dipolar coupling, it is likely to prove difficult to successfully execute uniform isotopic enrichment strategies to generate large numbers of quantitatively interpretable structural measurements in oriented sample NMR studies of membrane proteins. (ii) There are a number of readily implementable specific isotopic labeling schemes which can yield data patterns sufficient to identify local secondary structure for transmembrane segments of idealized proteins which are tilted by < 10 degrees with respect to the bilayer normal. (iii) The measurement of dipolar coupling constants between 13C-, 19F-, and/or 3H-labeled side chains of proximal residues may prove effective as routes to long range tertiary structural data constraints.