Effects of phytohaemagglutinin, wheat-germ agglutinin, and concanavalin-A on the physical state of sialic acid and membrane proteins in human erythrocyte ghosts: a spin label study

Parallel experiments employing sialic acid- and protein specific spin labels have been performed to monitor the effects on the physical state of this carbohydrate and membrane proteins of human erythrocytes induced by the binding of three lectins, $\text{Phaseolus vulgaris}$ phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), and Concanavalin-A (Con-A). PHA and WGA, both of which are known to bind at different sites on the principal sialoglycoprotein of human erythrocytes, glycophorin, had markedly different effects: compared to control values, PHA decreased the apparent correlation time of the sialic acid specific spin probe by 10% while this parameter was decreased by 33% by WGA. The protein specific spin label also monitored differential effects of these lectins: the relevant electron spin resonance parameter (the W/S ratio) was reduced 33% by PHA and increased by WGA over 17% from that of control values. Con-A, which is known to bind to the principal transmembrane protein, Band 3, had no effect on sialic acid or membrane proteins as assessed by the two spin labels employed. These results suggest that (1) the effects of binding of these different lectins, two of which bind to the same cell surface receptor, can be discriminated by use of spin labeling methods; (2) binding events occuring at the cell surface have distinct and pronounced effects on the physical state of proteins within the membrane; (3) the different results with PHA and WGA both of which bind to glycophorin are indicative of multiple and complex interactions of this glycoprotein with the membrane proteins in the erythrocyte; and (4) that the spin labelling technique has the potential to investigate the relationships between cell-surface binding events to membrane structural-functional interactions.     

A glycosynapse in myelin?

Myelin, the multilayered membrane which surrounds nerve axons, is the only example of a membranous structure where contact between extracellular surfaces of membrane from the same cell occurs. The two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I(3)-sulfate (SGC), can interact with each other by trans carbohydrate-carbohydrate interactions across apposed membranes. They occur in detergent-insoluble lipid rafts containing kinases and thus may be located in membrane signaling domains. These signaling domains may contact each other across apposed extracellular membranes, thus forming glycosynapses in myelin. Multivalent forms of these carbohydrates, GalC/SGC-containing liposomes, or galactose conjugated to albumin, have been added to cultured oligodendrocytes (OLs) to mimic interactions which might occur between these signaling domains when OL membranes or the extracellular surfaces of myelin come into contact. These interactions between multivalent carbohydrate and the OL membrane cause co-clustering or redistribution of myelin GSLs, GPI-linked proteins, several transmembrane proteins, and signaling proteins to the same membrane domains. They also cause depolymerization of the cytoskeleton, indicating that they cause transmission of a signal across the membrane. Their effects have similarities to those of anti-GSL antibodies on OLs, shown by others, suggesting that the multivalent carbohydrate interacts with GalC/SGC in the OL membrane. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature compact internodal myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.     

Effects of intramembrane particle aggregation on erythrocyte membrane fluidity: an electron spin resonance study in normal and in dystrophic subjects

Mobilization and aggregation of intramembrane particles (IMPs) are physiological events observed in various cells. In erythrocyte membranes, aggregation of IMPs can be induced by the exposure of partially desprectrinized erythrocyte membranes to acidic pH. We investigated the association between IMPs aggregation, protein mobility, and membrane fluidity in erythrocyte membranes of healthy controls and Duchenne muscular dystrophy (DMD) patients by using electron spin resonance and specific spin labels for membrane proteins and lipids. In erythrocyte membranes of control subjects, the partial spectrin removal induced a decreased segmental motion of protein spin label indicating an increase of protein-protein interactions. Stearic acid spin labels 5- and 16-(N-oxyl-4,4'-dimethyloxazolidine) showed that the treatment induces an increase of membrane fluidity. In DMD patients, both treated and untreated erythrocyte membranes showed changes of membrane fluidity when compared to those of the controls. Our results suggest that defects in the interactions between skeletal proteins and/or between membrane and skeleton components may contribute to the alterations of erythrocyte membranes in DMD.     

ESR studies of the erythrocyte membrane skeletal protein network: influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates

The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bisphosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P less than 0.002 and P less than 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding site is observed (P less than 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P less than 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.     

ESR studies of the erythrocyte membrane skeletal protein network: Influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates

The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bis-phosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P < 0.002 and P < 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding sites is observed (P < 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P < 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.     

Global structural changes in annexin 12. The roles of phospholipid,Ca2+, and pH

Ca2+-dependent membrane interaction has long been recognized as a general property of the annexin (ANX) family of proteins. More recently, it has become clear that ANXs can also undergo Ca2+-independent membrane interactions at mildly acidic pH. Here we use site-directed spin labeling in combination with circular dichroism and biochemical labeling methods to compare the structure and membrane topography of these two different membrane-bound forms of ANX12. Our results reveal strong similarities between the solution structure and the structure of the Ca2+-dependent membrane-bound form at neutral pH. In contrast, all Ca2+-independent membrane interactions tested resulted in large scale conformational changes and membrane insertion. Pairs of spin labels that were in close proximity across the interface of different domains of the protein in both the soluble and Ca2+-dependent membrane form were >25 A apart in the Ca2+-independent membrane-bound form. Despite these major conformational changes, the overall secondary structure content did not appear to be strongly altered and ANX12 remained largely helical. Thus, Ca2+-independent membrane interaction leads to massive refolding but not unfolding. Refolding did not occur at low pH in the absence of membranes but occurred within a few seconds after phospholipid vesicles were added. The phospholipid composition of the vesicles was an important modulator of Ca2+-independent membrane interaction. For example, cardiolipin-containing vesicles induced Ca2+-independent membrane interaction even at near neutral pH, thereby raising the possibility that lipid composition could induce relatively rapid Ca2+-independent membrane interaction in vivo.     

Structure determination of membrane proteins by NMR spectroscopy.

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.     

Innovative modular membrane adsorber system for high-throughput downstream screening for protein purification

To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8-strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8-strip anion exchange and the cation exchange membrane. The isolation of His(6)-tagged proteins, Bgl-His and GFP-His from fermentation broth and lysate, respectively, was performed using an 8-strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl-His) was studied in 8-strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu(2+) ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale-up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.     

In situ 19F NMR studies of an E. coli membrane protein

In this report, (19)F spin incorporation in a specific site of a specific membrane protein in E. coli was accomplished via trifluoromethyl-phenylalanine ((19) F-tfmF). Site-specific (19)F chemical shifts and longitudinal relaxation times of diacylglycerol kinase (DAGK), an E. coli membrane protein, were measured in its native membrane using in situ magic angle spinning (MAS) solid state nuclear magnetic resonance (NMR). Comparing with solution NMR data of the purified DAGK in detergent micelles, the in situ MAS-NMR data illustrated that (19)F chemical shift values of residues at different membrane protein locations were influenced by interactions between membrane proteins and their surrounding lipid or lipid mimic environments, while (19)F side chain longitudinal relaxation values were probably affected by different interactions of DAGK with planar lipid bilayer versus globular detergent micelles.     

Epigenetic effectiveness of complete carcinogens: specific interactions of polycyclic aromatic hydrocarbons and aminoazo dyes with cholesterol and apolipoprotein A-I

During a co-precipitation of cholesterol (Chol) and slight amounts of polycyclic aromatic hydrocarbons (PAHs) or aminoazo dyes (AZOs) in aqueous albumin solution, complex particles are formed; on their surfaces apolipoproteins with an amphipathic alpha-helix (e.g. apoA-I) are more or less firmly adsorbed. An efficacy index can be calculated from the strength of the hydrophobic interactions between apoA-I and the [Chol/PAH]- or [Chol/AZO]-complex, and the solubility of the PAH or AZO in an aqueous medium, which correlates to the carcinogenicity of these compounds. A short-term test for PAHs and AZOs is described, in which the efficacy index can be determined in the simplest manner without any great expenditure on equipment. The previous results suggest that the parent compounds of the PAHs and AZOs can be involved in a specific interaction with cholesterol-domains of the plasma membrane of a cell. The changes in membrane fluidity and architecture caused by these specific interactions could modulate the distribution and/or activity of membrane proteins which are critical to the regulation of cellular proliferation.     

Structure and dynamics of the phospholipase C-delta1 pleckstrin homology domain located at the lipid bilayer surface

Despite the importance of signal transduction pathways at membrane surfaces, there have been few means of investigating their molecular mechanisms based on the structural information of membrane-bound proteins. We applied solid state NMR as a novel method to obtain structural information about the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain at the lipid bilayer surface. NMR spectra of the alanine residues in the vicinity of the beta5/beta6 loop in the PH domain revealed changes in local conformations due to the membrane localization of the protein. We propose that these conformational changes originate from a hydrophobic interaction between the amphipathic alpha-helix located in the beta5/beta6 loop and the hydrophobic layer of the membrane and contribute to the membrane binding affinity, interdomain interactions and intermolecular interactions of PLC-delta1.     

Defining Specific Lipid Binding Sites for a Peripheral Membrane Protein in Situ Using Subtesla Field-cycling NMR

Despite the profound physiological consequences associated with peripheral membrane protein localization, only a rudimentary understanding of the interactions of proteins with membrane surfaces exists because these questions are inaccessible by commonly used structural techniques. Here, we combine high resolution field-cycling ³¹P NMR relaxation methods with spin-labeled proteins to delineate specific interactions of a bacterial phospholipase C with phospholipid vesicles. Unexpectedly, discrete binding sites for both a substrate analogue and a different phospholipid (phosphatidylcholine) known to activate the enzyme are observed. The lifetimes for the occupation of these sites (when the protein is anchored transiently to the membrane) are >1–2 μs (but <1 ms), which represents the first estimate of an off-rate for a lipid dissociating from a specific site on the protein and returning to the bilayer. Furthermore, analyses of the spin-label induced NMR relaxation corroborates the presence of a discrete tyrosine-rich phosphatidylcholine binding site whose location is consistent with that suggested by modeling studies. The methodology illustrated here may be extended to a wide range of peripheral membrane proteins.     

Computational analysis of the membrane association of group IIA secreted phospholipases A2: a differential role for electrostatics

Secreted phospholipases A2 (sPLA2's) are enzymes that hydrolyze glycerophospholipids at the sn-2 position, which leads to the production of lipid mediators of many cellular processes. These interfacial enzymes are regulated by their lipid specificity at two levels: membrane binding and substrate recognition. Different sPLA2's utilize different combinations of electrostatic and hydrophobic interactions to adsorb to membrane surfaces, which results in the wide range of membrane binding behaviors observed. Here, the finite difference Poisson Boltzmann (FDPB) method is used to quantitatively analyze the contribution of electrostatic interactions to the membrane association of two highly basic group II sPLA2's: Agkistrodon piscivorus piscivorus (AppD49) sPLA2 and nonpancreatic human group IIA (hGIIA) sPLA2. The calculations predict how membrane binding is affected by ionic strength, membrane composition, substitutions of residues in the enzymes, and the presence of calcium in the active site. In addition, the results provide molecular models for the membrane-associated forms of the enzymes. Furthermore, these models account for (1) changes in orientation and protonation state of both the native and charge reversal forms of the enzymes at the membrane surface and (2) the effect of protein/vesicle aggregation, as observed for hGIIA sPLA2. Importantly, the modeling quantitatively describes the complex membrane binding behaviors of these interfacial enzymes in terms of simple physical forces and provides structural information that is difficult to obtain experimentally. The computational analysis shows that nonspecific electrostatic interactions not only play a major role in recruiting these enzymes to membrane surfaces but also orient the enzymes for productive catalysis at the membrane interface.     

Quinolinic acid, an endogenous metabolite with neurotoxic properties, alters the physical state of membrane proteins in human erythrocytes

Quinolinic acid (2,3-pyridinedicarboxylic acid), an endogenous, tryptophan metabolite, is neurotoxic when injected into rat striatum (1). To begin to investigate the molecular interactions of quinolinic acid with membranes, electron spin resonance studies of the effects of this neurotoxin on the physical state of lipids, proteins, and cell-surface sialic acid in human erythrocyte ghosts have been performed. Quinolinic acid induced a highly significant alteration in the physical state of membrane proteins (P less than 0.01) while that of sialic acid and membrane lipids was unaffected. These results are similar to those induced by ibotenic acid, an exogenous neurotoxin, and are discussed with reference to possible molecular characteristics of the interaction of these neurotoxins with membrane proteins.     

The T cell receptor triggering apparatus is composed of monovalent or monomeric proteins

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.     

Lipid-protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy

Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.     

Interaction Between Equally Charged Membrane Surfaces Mediated by Positively and Negatively Charged Macro-Ions

In biological systems, charged membrane surfaces are surrounded by charged molecules such as electrolyte ions and proteins. Our recent experiments in the systems of giant phospholipid vesicles indicated that some of the blood plasma proteins (macro-ions) may promote adhesion between equally charged membrane surfaces. In this work, theory was put forward to describe an IgG antibody-mediated attractive interaction between negatively charged membrane surfaces which was observed in experiments on giant phospholipid vesicles with cardiolipin-containing membranes. The attractive interactions between negatively charged membrane surfaces in the presence of negatively and positively charged spherical macro-ions are explained using functional density theory and Monte Carlo simulations. Both, the rigorous solution of the variational problem within the functional density theory and the Monte Carlo simulations show that spatial and orientational ordering of macro-ions may give rise to an attractive interaction between negatively charged membrane surfaces. It is also shown that the distinctive spatial distribution of the charge within the macro-ions (proteins) is essential in this process.     

Interhelical hydrogen bonding drives strong interactions in membrane proteins

Polar residues in transmembrane alpha-helices may strongly influence the folding or association of integral membrane proteins. To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper. We found in both detergent micelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence. Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur. Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces.     

Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.