Tyrosine fluorescence analysis of apolipophorin III-lipopolysaccharide interaction

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein that binds to lipopolysaccharides (LPS). Polyacrylamide gel electrophoresis analysis demonstrated that apoLp-III from Galleria mellonella associated with various truncated LPS variants, including lipid A. Subsequent binding studies were performed employing the intrinsic tyrosine fluorescence properties of apoLp-III, which is highly quenched in the unbound state. A marked increase in tyrosine fluorescence intensity was observed upon binding to LPS or detoxified LPS, indicating a new microenvironment for Tyr-142. This also implies that the LPS carbohydrate region is involved in LPS binding. Dissociation constants (Kd) measured by apoLp-III titration were estimated at approximately 1 microM. Increasing the ionic strength did not decrease the Kd, neither did LPS phosphate removal. In addition, truncation apoLp-III mutants, lacking two complete helices, were still able to associate with LPS. This indicates that the association of apoLp-III with LPS may not be governed by charge but by hydrophobic interactions.     

Apolipophorin III lysine modification: Effect on structure and lipid binding

Apolipophorin III (apoLp-III) from Locusta migratoria was used as a model to investigate apolipoprotein lipid binding interactions. ApoLp-III contains eight lysine residues, of which seven are located on one side of the protein. To investigate the role of positive charges on lipid binding, lysine residues were acetylated by acetic anhydride. The degree of acetylation was analyzed by SDS-PAGE and MALDI-TOF, indicating a maximum of eight acetyl additions. Modified apoLp-III remained α-helical, but displayed a decreased α-helical content (from 78 to 54%). Acetylation resulted in a slight increase in protein stability, as indicated by a change in the midpoint of guanidine-HCl induced denaturation from 0.55 (unmodified) to 0.65 M (acetylated apoLp-III). Lipid bound apoLp-III, either acetylated or unmodified, displayed similar increases in helical content and midpoint of guanidine-HCl-induced denaturation of ∼ 4 M. The ability to solubilize vesicles of dimyristoylphosphatidylcholine remained unchanged. However, the rate to solubilize dimyristoylphosphatidylglycerol vesicles was reduced two-fold. In addition, a decreased ability to stabilize diacylglycerol-enriched low density lipoproteins was observed. This indicated that lysine residues are not critical for the protein's ability to bind to zwitterionic phospholipids. Since binding interactions with ionic phospholipids and lipoproteins were affected by acetylation, lysine side-chains may play a modulating role in the interaction with more complex lipid surfaces encountered in vivo.     

An N-terminal three-helix fragment of the exchangeable insect apolipoprotein apolipophorin III conserves the lipid binding properties of wild-type protein

Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.     

Apolipophorin III: lipopolysaccharide binding requires helix bundle opening

Apolipophorin III (apoLp-III) is a prototypical apolipoprotein used for structure-function studies. Besides its crucial role in lipid transport, apoLp-III is able to associate with fungal and bacterial membranes and stimulate cellular immune responses. We recently demonstrated binding interaction of apoLp-III of the greater wax moth, Galleria mellonella, with lipopolysaccharides (LPS). In the present study, the requirement of helix bundle opening for LPS binding interaction was investigated. Using site-directed mutagenesis, two cysteine residues were introduced in close spatial proximity (P5C/A135C). When the helix bundle was locked by disulfide bond formation, the tethered helix bundle failed to associate with LPS. In contrast, the mutant protein regained its ability to bind upon reduction with dithiothreitol. Thus, helix bundle opening is a critical event in apoLp-III binding interaction with LPS. This mechanism implies that the hydrophobic interior of the protein interacts directly with LPS, analogous to that observed for lipid interaction.     

Lipopolysaccharide binding of an exchangeable apolipoprotein, apolipophorin III, from Galleria mellonella

A new role of apolipophorin III (apoLp-III) as an immune activator has emerged recently. To gain insight into this novel function, the interaction of apoLp-III with lipopoly-saccharide (LPS) was investigated. ApoLp-III from Galleria mellonella was incubated with LPS from Escherichia coli O55:B5, and analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Protein staining showed that apoLp-III mobility was significantly reduced. In addition, silver and LPS fluorescent staining demonstrated that LPS mobility was increased upon incubation with apoLp-III. This result suggests association of apoLp-III with LPS. Sodium dodecyl sulfate (SDS) PAGE analysis showed decreased apoLp-III mobility upon LPS addition, indicative of LPS apoLp-III interaction in the presence of SDS. The unique tyrosine residue that resides in apoLp-III was used to provide additional evidence for LPS binding interaction. In the absence of LPS, apoLp-III tyrosine fluorescence was relatively low. However, LPS addition resulted in a progressive increase in the fluorescence intensity, indicating tertiary rearrangement in the environment of tyrosine 142 upon LPS interaction. Other well-characterized apoLp-IIIs were also examined for LPS binding. Manduca sexta , Bombyx mori and Locusta migratoria apoLp-III were all able to interact with LPS. The ability of apoLp-III to form complexes with LPS supports the proposed role of apoLp-III in innate immunity.     

Interaction of an exchangeable apolipoprotein with phospholipid vesicles and lipoprotein particles. Role of leucines 32, 34, and 95 in Locusta migratoria apolipophorin III.

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipid surfaces. In the lipid-free state this 164-residue protein exists as a bundle of five elongated amphipathic alpha-helices. Upon lipid binding, apoLp-III undergoes a significant conformational change, resulting in exposure of its hydrophobic interior to the lipid environment. On the basis of x-ray crystallographic data (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), it was proposed that hydrophobic residues, present in loops that connect helices 1 and 2 (Leu-32 and Leu-34) and helices 3 and 4 (Leu-95), may function in initiation of lipid binding. To examine this hypothesis, mutant apoLp-IIIs were designed wherein the three Leu residues were replaced by Arg, individually or together. Circular dichroism spectroscopy and temperature and guanidine hydrochloride denaturation studies showed that the mutations did not cause major changes in secondary structure content or stability. In lipid binding assays, addition of apoLp-III to phospholipid vesicles caused a rapid clearance of vesicle turbidity due to transformation to discoidal complexes. L34R and L32R/L34R/L95R apoLp-IIIs displayed a much stronger interaction with lipid vesicles than wild-type apoLp-III. Furthermore, it was demonstrated that the mutant apoLp-IIIs retained their ability to bind to lipoprotein particles. However, in lipoprotein competition binding assays, the mutants displayed an impaired ability to initiate a binding interaction when compared with wild-type apoLp-III. The data indicate that the loops connecting helices 1 and 2 and helices 3 and 4 are critical regions in the protein, contributing to recognition of hydrophobic defects on lipoprotein surfaces by apoLp-III.     

Galleria mellonella apolipophorin III – an apolipoprotein with anti-Legionella pneumophila activity

The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III.     

Role of buried polar residues in helix bundle stability and lipid binding of apolipophorin III: destabilization by threonine 31

Apolipophorin III (apoLp-III) from Locusta migratoria is a model exchangeable apolipoprotein that plays a key role in neutral lipid transport. The protein is comprised of a bundle of five amphipathic alpha-helices, with most hydrophobic residues buried in the protein interior. The low stability of apoLp-III is thought to be crucial for lipid-induced helix bundle opening, to allow protein-lipid interactions. The presence of polar residues in the hydrophobic protein interior may facilitate this role. To test this, two buried polar residues, Thr-31 and Thr-144, were changed into alanine by site-directed mutagenesis. Secondary structure analysis and GdnHCl- and temperature-induced denaturation studies indicated an increase in alpha-helical content and protein stability for T31A apoLp-III compared to wild-type apoLp-III. In contrast, T144A had a decreased alpha-helical content and protein stability, while tryptophan fluorescence indicated increased exposure of the hydrophobic interior to buffer. Two mutant proteins that had lysine residues introduced in the hydrophobic core displayed a more pronounced decrease in secondary structure and protein stability. Lipid binding studies using phospholipid vesicles showed that T31A apoLp-III was able to transform phospholipid vesicles into discoidal particles but at a 3-fold reduced rate compared to wild-type apoLp-III. In contrast, the less stable apoLp-III mutants displayed an increased ability to transform phospholipid vesicles. These results demonstrate the inverse correlation between protein stability and the ability to transform phospholipid vesicles into discoidal protein-lipid complexes and that Thr-31 is a key determinant of the relatively low protein stability, thereby promoting apoLp-III to interact with lipid surfaces.     

Spectroscopic characterization of the conformational adaptability of Bombyx mori apolipophorin III.

Apolipophorin III (apoLp-III) from the silkmoth, Bombyx mori, has been over-expressed in Escherichia coli, purified and characterized. Far-UV CD spectroscopic analysis revealed 65% alpha-helix secondary structure. Near-UV CD spectra obtained in buffer or complexed with dimyristoylglycerophosphocholine (DMPC), provided evidence that apoLp-III alpha-helices reorient upon interaction with lipid, indicative of a protein conformational change. In guanidine hydrochloride (GdnHCl) denaturation studies, a transition midpoint of 0.33 M was observed, corresponding to a DeltaGDH2O = 2.46 kcal. mol-1. Fluorescence studies of the sole tryptophan residue (Trp40) in apoLp-III revealed an emission lambdamax = 327 nm. Compared to free tryptophan, Stern-Volmer constants (KSV) for acrylamide and KI quenching of Trp40 fluorescence were decreased by 20-fold and sevenfold, respectively. In studies of apoLp-III-DMPC disc complexes, far-UV CD spectroscopy revealed an increase in alpha-helix content to approximately 85% and a ninefold increase in the GdnHCl-induced denaturation transition midpoint to 3 M. In studies of lipid interaction, apoLp-III was shown to disrupt both negatively charged and zwitterionic phospholipid bilayer vesicles, transforming them into discoidal complexes. Characterization of apoLp-III-DMPC discs, using 5-doxyl or 12-doxyl stearic acid as lipid-based quenching agents, revealed that Trp40 localizes near the phospholipid polar head groups. KSV values for acrylamide and KI quenching of intrinsic fluorescence of apoLp-III-DMPC discs indicate that Trp40 is embedded in the lipid milieu, with little or no accessibility to the aqueous quenchers. Given the large amount of alpha-helix in apoLp-III, the data presented support a model in which amphipathic alpha-helical segments are stabilized by helix-helix interactions and lipid association induces a protein conformational change which results in substitution of helix-helix interactions for helix-lipid contacts.     

Structure-guided protein engineering modulates helix bundle exchangeable apolipoprotein properties

Apolipoprotein (apo) E plays a major role in lipid metabolism by mediating cellular uptake of lipoprotein particles through interaction with members of the low density lipoprotein (LDL) receptor family. The primary region of apoE responsible for receptor binding has been limited to a cluster of basic amino acids between residues 134 and 150, located in the fourth helix of the N-terminal domain globular helix bundle structure. To investigate structural and functional requirements of this "receptor binding region" we engineered an apolipoprotein chimera wherein residues 131-151 of human apoE were substituted for residues 146-166 (helix 5) of Manduca sexta apolipophorin III (apoLp-III). Recombinant hybrid apolipoprotein was expressed in Escherichia coli, isolated, and characterized. Hybrid apolipoprotein and apoE3-N-terminal, but not apoLp-III, bound to heparin-Sepharose. Far UV circular dichroism spectroscopy revealed the presence of predominantly alpha-helix secondary structure, and stability studies revealed a urea denaturation midpoint of 1.05 m, similar to wild-type apoLp-III. Hybrid apolipoprotein-induced dimyristoylphosphatidylcholine (DMPC) bilayer vesicle solubilization activity was significantly enhanced compared with either parent protein, consistent with detection of solvent-exposed hydrophobic regions on the protein in fluorescent dye binding experiments. Unlike wild-type apoLp-III.DMPC complexes, disc particles bearing the hybrid apolipoprotein competed with 125ILDL for binding to the LDL receptor on cultured human skin fibroblasts. We conclude that a hybrid apolipoprotein containing a key receptor recognition element of apoE preserves the structural integrity of the parent protein while conferring a new biological activity, illustrating the potential of helix swapping to introduce desirable biological properties into unrelated or engineered apolipoproteins.     

Interaction of locust apolipophorin III with lipoproteins and phospholipid vesicles: effect of glycosylation

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipoprotein surfaces. The native protein is glycosylated at Asn-18 and Asn-85. Variable attachment of five distinct oligosaccharide moieties at the two glycosylation sites results in molecular weight heterogeneity, as seen by mass spectrometry. The main mass peak of 20,488 Da decreases to 17,583 Da after removal of carbohydrate, indicating that apoLp-III carbohydrate mass is approximately 14% by weight. Deglycosylated apoLp-III induced clearance of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol vesicles at a faster rate than glycosylated apoLp-III. However, in lipoprotein binding assays, in which apoLp-III interacts with surface-localized diacylglycerol, only minor differences in binding were observed. The fluorescence properties of 1-anilinonaphthalene-8-sulfonate were unaffected by the glycosylation state of apoLp-III, indicating that no changes in the relative amount of exposed hydrophobic surface occurred as a result of carbohydrate removal. We propose that glycosyl moieties affect the ability of apoLp-III to transform phospholipid bilayer vesicles into disc-like complexes by steric hindrance. This is due to the requirement that apoLp-III penetrate the bilayer substrate prior to conformational opening of the helix bundle. On the other hand, the glycosyl moieties do not affect lipoprotein binding interactions as it does not involve deep protein penetration into the lipid milieu. Rather, lipoprotein binding is based on oriented protein contact with the lipid surface followed by opening of the helix bundle, which allows formation of a stable interaction with surface exposed hydrophobic sites.     

Simultaneous Preparation and Quantitation of Proteoglycans by Precipitation with Alcian Blue

Conditions for specific interaction between Alcian blue and proteoglycans were optimized by comparing the differential spectra of Alcian blue obtained with purified chondroitin sulfate dissolved in water with the spectra obtained with nasal cartilage proteoglycans dissolved in synovial fluid. A method was then designed that provides specific precipitation of proteoglycans or glycosaminoglycans in 4 M guanidine-HCl in the presence of protein, hyaluronic acid, or nucleic acids. The specificity is achieved by using a low pH in combination with detergent and high salt concentration. Stepwise addition of reagents is necessary to avoid binding of Alcian blue to proteins and nucleic acids. All polyanions, except polysulfates, are first neutralized by lowering the pH to 1.5. By including detergent in this step, the hydrophobic protein regions are blocked and not accessible for binding with the dye. These regions could otherwise bind Alcian blue by hydrophobic interaction. When the Alcian blue reagent is added after, only the polysulfated molecules will remain charged and free to interact with Alcian blue. At least 0.4 M guanidine-HCl is required to abolish the negative interference by proteins. All sulfated glycosaminoglycans are precipitated at 0.4 M guanidine-HCl. With increasing guanidine-HCl concentrations, the different glycosaminoglycans are precipitated in accordance with the critical electrolyte concentration of the respective glycosaminoglycan. The Alcian blue precipitation can be performed at different concentrations of guanidine-HCl in order to separate different classes of proteoglycans. Excess dye and contaminating proteins are removed by a wash in a DMSO-MgCl₂ solution and the precipitate is dissolved in a mixture of guanidine-HCl and propanol. For quantitation, the absorbance is recorded in a microplate reader with the 600-nm filter, the assay being linear between 0.5 and 20 μg proteoglycan. Since no digestion of samples with protease is needed, the proteoglycans are recovered in native form. The proteoglycan-Alcian blue complexes dissociate in the guanidine-HCl/propanol mixture and the proteoglycans can be selectivelyprecipitated with propanol. The dye is used for quantitation and the proteoglycans can be utilized for further analysis.     

Insect immune activation by apolipophorin III is correlated with the lipid-binding properties of this protein

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.     

A novel role for an insect apolipoprotein (apolipophorin III) in beta-1,3-glucan pattern recognition and cellular encapsulation reactions

Lipoproteins and molecules for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study we describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide production in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study we show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and beta-1,3-glucan and therefore may act as a pattern recognition molecule for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiological levels, and, with the notable exception of beta-glucan binding, are only observed with the lipid-associated form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition molecule and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunology, particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.     

Studies on aspartase. IV. Reversible denaturation of Escherichia coli aspartase.

Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) of Escherichia coli, denatured in 4 M guanidine-HCl, was renatured in vitro by simple dilution with a concomitant restoration of the activity. While the native enzyme exhibited a marked negative Cotton effect centered at 233 +/- 1 nm in optical rotatory dispersion, the enzyme denatured in 4 M guanidine-HCl retained little optical activity. Upon dilution of the denatured enzyme, however, more than 90% of the ordered structure was recovered in 1 min, while the restoration of the activity proceeded much more slowly. Estimation of molecular weights by gel permeation chromatography indicated that the tetrameric enzyme is subject to reversible dissociation into monomeric subunits under the experimental conditions. Various environmental factors such as temperature, pH and protein concentration exhibited profound influence on the rate and extent of the reactivation. In order to examine the correlation between the restoration of the activity and the quaternary structure, electron microscopic inspection of the kinetic processes of reversible denaturation was attempted. Upon dilution of the denatured enzyme at 4 degrees C, neither the activity nor tetrameric images were detected over several min. Upon the temperature shift up to 25 degrees C, however, the activity regain was rapidly proceeded concomitant with the appearance of tetrameric molecules. These results are compatible with the possibility that the subunit assembly is an essential prerequisite, thought not sufficient, for enzyme activity.     

Apolipophorin III interaction with model membranes composed of phosphatidylcholine and sphingomyelin using differential scanning calorimetry

Apolipophorin III (apoLp-III) from Locusta migratoria was employed as a model apolipoprotein to gain insight into binding interactions with lipid vesicles. Differential scanning calorimetry (DSC) was used to measure the binding interaction of apoLp-III with liposomes composed of mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SM). Association of apoLp-III with multilamellar liposomes occurred over a temperature range around the liquid crystalline phase transition (L_α). Qualitative and quantitative data were obtained from changes in the lipid phase transition upon addition of apoLp-III. Eleven ratios of DMPC and SM were tested from pure DMPC to pure SM. Broadness of the phase transition (T_1/2), melting temperature of the phase transition (T_m) and enthalpy were used to determine the relative binding affinity to the liposomes. Multilamellar vesicles composed of 40% DMPC and 60% SM showed the greatest interaction with apoLp-III, indicated by large T_1/2 values. Pure DMPC showed the weakest interaction and liposomes with lower percentage of DMPC retained domains of pure DMPC, even upon apoLp-III binding indicating demixing of liposome lipids. Addition of apoLp-III to rehydrated liposomes was compared to codissolved trials, in which lipids were rehydrated in the presence of protein, forcing the protein to interact with the lipid system. Similar trends between the codissolved and non-codissolved trials were observed, indicating a similar binding affinity except for pure DMPC. These results suggested that surface defects due to non-ideal packing that occur at the phase transition temperature of the lipid mixtures are responsible for apolipoprotein-lipid interaction in DMPC/SM liposomes.     

Effects of denaturants on the sweet-tasting protein monellin.

Effects of the denaturants urea and guanidine-HCl on the sweet-tasting protein monellin have been studied. The pH at which monellin is initially treated with denaturant is an important factor in retention of sweetness, but the pH maintained during subsequent removal of denaturant by dialysis has no effect on activity. Recovery of sweetness of denaturant-treated monellin is favored when denaturation occurs at acid pH. Monellin treated with either 6 M guanidine-HCl or 8 M urea at acid pH retains all of its sweetness following removal of denaturant, but urea treatment at neutral pH leads to some irreversible loss of sweetness. Monellin precipitates from solution under some conditions during removal of denaturant by dialysis, and the precipitated protein is no longer sweet. Precipitation is least under acid conditions. Aggregated protein was demonstrated by gel filtration chromatography. The single sulfhydryl group of monellin was not demonstrable in the precipitated protein, having apparently become oxidized during denaturation and formation of the aggregated protein. The data support the hypothesis that the tertiary structure is important in the ability of monellin to elicit a sweet sensation.     

Investigation of spectrin binding to phospholipid vesicles using isoindole fluorescent probe. Thermal properties of the bound and unbound protein

Fluorescence of isoindole probe covalently bound to spectrin from pig erythrocytes, and fluorescence of tryptophanyl residues were used to study spectrin interaction with phospholipid bilayers. Evidence would be provided for conformational changes of spectrin occurring upon its binding to lipid bilayers. Fluorescence quenching experiments allowed to determine thermal stability of the protein in bound and unbound state. Spectrin binding to lipids was shown to protect the protein against thermal denaturation.     

Apolipophorin III: a lipid-triggered molecular switch

Apolipophorin III (apoLp-III) is a low molecular weight exchangeable apolipoprotein that plays an important role in the enhanced neutral lipid transport during insect flight. The protein exists in lipid-free and lipid-bound states. The lipid-bound state is the active form of the protein and occurs when apoLp-III associates with lipid-enriched lipophorins. ApoLp-III is well characterized in two evolutionally divergent species: Locusta migratoria and Manduca sexta. The two apolipoproteins interact in a similar manner with model phospholipid vesicles, and transform them into discoidal particles. Their low intrinsic stability in the lipid-free state likely facilitates interaction with lipid surfaces. Low solution pH also favors lipid binding interaction through increased exposure of hydrophobic surfaces on apoLp-III. While secondary structure is maintained under acidic conditions, apoLp-III tertiary structure is altered, adopting molten globule-like characteristics. In studies of apoLp-III interaction with natural lipoproteins, we found that apoLp-III is readily displaced from the surface of L. migratoria low-density lipophorin by recombinant apoLp-III proteins from either L. migratoria or M. sexta. Thus, despite important differences between these two apoLp-IIIs (amino acid sequence, presence of carbohydrate), their functional similarity is striking. This similarity is also illustrated by the recently published NMR solution structure of M. sexta apoLp-III wherein its molecular architecture closely parallels that of L. migratoria apoLp-III.     

Differential lipid binding of truncation mutants of Galleria mellonella apolipophorin III

Apolipophorin III (apoLp-III) is a prototype exchangeable apolipoprotein that is amenable to structure-function studies. The protein folds as a bundle of five amphipathic alpha-helices and undergoes a dramatic conformational change upon lipid binding. Recently, we have shown that a truncation mutant of Galleria mellonella apoLp-III comprising helices 1-3 is stable in solution and able to bind to lipid surfaces [Dettloff, M., Weers, P. M. M., Niere, M., Kay, C. M., Ryan, R. O., and Wiesner, A. (2001) Biochemistry 40, 3150-3157]. To investigate the role of the C-terminal helices in apoLp-III structure and function, two additional 3-helix mutants were designed: a core fragment comprising helix (H) 2-4, and a C-terminal fragment (H3-5). Each truncation mutant retained the ability to associate spontaneously with dimyristoylphosphatidylcholine (DMPC) vesicles, transforming them into discoidal complexes. The rate of apolipoprotein-dependent DMPC vesicle transformation decreased in the order H1-3 > H2-4 > H3-5. Truncation of two helices led to a significant decrease in alpha-helical content in buffer in each case, from 86% (wild-type) to 50% (H1-3), 28% (H2-4), and 24% alpha-helical content (H3-5). On the other hand, trifluoroethanol or complexation with DMPC induced the truncation mutants to adopt a high alpha-helical structure similar to that of wild-type protein (84-100% alpha-helical structure). ApoLp-III(H1-3) and apoLp-III(H2-4), but not apoLp-III(H3-5), were able to prevent phospholipase-C-induced low density lipoprotein aggregation, indicating that interaction of the C-terminal fragment with spherical lipoprotein surfaces was impaired. As lipoprotein binding is significantly affected and DMPC transformation rates are relatively slow upon removal of N-terminal helices, the data indicate that structural elements necessary for lipid interaction reside in the N-terminal part of the protein.