Cholesterol-induced alteration of apolipoprotein A-I conformation in reassembled high density lipoprotein.

Recent reports have shown that apolipoprotein A-I (apo A-I), the major protein of high density lipoprotein (HDL) may exist in different conformational states. We studied the effects of apolipoprotein A-II and/or cholesterol on the conformation of apo A-I in reassembled HDL. Analysis of tryptophan fluorescence quenching in the presence of iodine suggested that cholesterol increased the number of apo A-I tryptophan residues accessible to the aqueous phase, but decreased their mean degree of hydration. These observations cannot be totally explained on the basis of the effect of cholesterol on phospholipid viscosity as determined by fluorescence anisotropy of diphenyl hexatriene. We did not observe any effect of apo A-II on the conformation of apo A-I.     

Spectroscopic studies of the tyrosine residues of human plasma apolipoprotein A-II

Human apolipoprotein A-II (apo A-II) in solution and associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was investigated by a combination of absorbance and fluorescence methods. Each apo A-II polypeptide chain contains four tyrosine residues but no tryptophan residues. Two and three tyrosine residues, respectively, appear to be buried for apo A-II in aqueous solution and in the lipid-associated protein. The spectroscopic properties of the tyrosine residues of lipid-associated apo A-II were also investigated. Plots of fluorescence intensity against temperature revealed a discontinuity in the region of the phase transition; however, over the same temperature range, there was no change in the exposure of tyrosine residues to the aqueous environment or in their mobility as measured by fluorescence polarization. Near-ultraviolet circular dichroic measurements demonstrated that the environments of the tyrosine residues of lipid-associated apo A-II and nitrated apo A-II were different from that of the apo A-II in solution or in a denatured state. Similar measurements also revealed that the microenvironments around tyrosines of apo A-II bound to DMPC in the gel phase are different from those observed in the liquid crystalline phase. Using environmentally sensitive fluorescence lipid probes, we have previously demonstrated that the polarity of the lipid/water interface of DMPC changes through a phase transition. The observations presented here indicate that these environmental changes also occur at the lipid/protein interface.     

Mechanism of dissociation of human apolipoprotein A-I from complexes with dimyristoylphosphatidylcholine as studied by guanidine hydrochloride denaturation

The reversibility of the binding of human apolipoprotein A-I (apo A-I) to phospholipid has been monitored through the influence of guanidine hydrochloride (Gdn-HCl) on the isothermal denaturation and renaturation of apo A-1/dimyristoylphosphatidylcholine (DMPC) complexes at 24 degree C. Denaturation was studied by incubating discoidal 1:100 and vesicular 1:500 mol/mol apo A-I/DMPC complexes with up to 7 M Gdn-HCl for up to 72 h. Unfolding of apo A-I molecules was observed from circular dichroism spectra while the distribution of protein between free and lipid-associated states was monitored by density gradient ultracentrifugation. The ability of apo A-I to combine with DMPC in the presence of Gdn-HCl at 24 degrees C was also investigated by similar procedures. In both the denaturation and renaturation of 1:100 and 1:500 complexes, the final values of the molar ellipticity and the ratio of free to bound apo A-I at various concentrations of Gdn-HCl are dependent on the initial state of the lipid and protein; apo A-I is more resistant to denaturation when Gdn-HCl is added to existing complexes than to a mixture of apo A-I and DMPC. There is an intermediate state in the denaturation pathway of apo A-I/DMPC complexes which is not present in the renaturation; the intermediate comprises partially unfold apo A-I molecules still associated with the complex by some of their apolar residues. Complete unfolding of the alpha helix and subsequent desorption of the apo A-I molecules from the lipid/water interface involve cooperative exposure of these apolar residues to the aqueous phase. The energy barrier associated with this desorption step makes the binding of apo A-I to DMPC a thermodynamically irreversible process. Consequently, binding constants of apo A-I and PC cannot be calculated simply from equilibrium thermodynamic treatments of the partitioning of protein between free and bound states. Apo A-I molecules do not exchange freely between the lipid-free and lipid-bound states, and extra work is required to drive protein molecules off the surface. The required increased in surface pressure can be achieved by a net mass transfer of protein to the surface; in vivo, increases in the surface pressure of lipoproteins by lipolysis can cause protein desorption.     

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: a study by oxygen quenching and fluorescence depolarization

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 degrees C as compared to that at 5 degrees C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC complexes.     

Non enzymatic glycation of apolipoprotein A-I. Effects on its self-association and lipid binding properties

In diabetic patients, hyperglycaemia results in the non enzymatic glycation of many proteins including apolipoprotein A-I. We purified glycated apo A-I and compared its lipid binding properties to those of normal apo A-I. Analysis of tryptophan fluorescence spectra and of fluorescence quenching in the presence of iodine showed that glycation of apo A-I induces a decrease in the stability of the lipid-apoprotein interaction and in that of the apoprotein self-association. Repetitive ultracentrifugations of High Density Lipoprotein (HDL) samples containing radioiodinated apo A-I or glycated apo A-I revealed that glycation of the apoprotein facilitates its dissociation from HDL. These results suggest that the non enzymatic glycation of apo A-I may affect the structural cohesion of HDL particles.     

Tryptophan fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers

The tryptophan intrinsic fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers was examined at different temperatures. Absorption and emission maxima occur at 277 and 332 nm, irrespective of temperature or lipid:protein ratio even if there are indications (from fluorescence quenching) of protein conformational changes as a function of lipid:protein ratio. Low values of Trp fluorescence quantum yield in complex III (0.008-0.010) are probably due to the neighborhood of the heme groups. The temperature-dependent decrease of fluorescence intensity is nonlinear; the corresponding Arrhenius plots show "breaks" or discontinuities that could be interpreted as thermally dependent changes in protein conformation. However, no temperature-dependent changes in fluorescence quenching have been observed that may be related to protein conformational changes. In addition, Arrhenius plots of the fluorescence intensity of simple molecules, such as Trp or 1-anilino-8-naphthalene sulfonate in the presence of aqueous phospholipid dispersions, also show breaks in the same temperature range. Stern-Volmer plots of acrylamide and iodide quenching were also nonlinear, indicating large differences in quenching constants for the various tryptophanyl residues. The quenching results also suggest that, at high lipid:protein ratios, the microviscosity of the protein matrix is higher than that in lipid-poor systems. Comparison of quenching efficiencies of iodide and acrylamide suggest that no significant fraction of the fluorophores occurs in the neighborhood of charged residues.     

Human apolipoprotein A-I forms thermally stable complexes with anionic but not with zwitterionic phospholipids

The mechanism of the association of human plasma apolipoprotein A-I (apo A-I) with the acidic phospholipids, dimyristoylphosphatidylglycerol (DMPG), egg yolk phosphatidylglycerol, and dioleoylphosphatidylserine as well as with the zwitterionic dimyristoylphosphatidylcholine (DMPC) has been studied using turbidimetry, circular dichroism, high-sensitivity differential scanning calorimetry, and electron microscopy. The association of apo A-I with multilamellar liposomes of acidic phospholipids is rapid over a broad temperature range at and above the temperature of the lipid gel to liquid crystalline transition, Tc. This is in contrast to zwitterionic phosphatidylcholine which recombines with apo A-I only over a narrow temperature range around Tc. The complex of apo A-I with DMPC denatures at elevated temperatures giving rise to a calorimetrically detectable transition. The temperature range and width of this transition is shown to be markedly dependent on the heating rate. This is again in contrast to apo A-I recombinants with DMPG which show no calorimetrically detectable thermal denaturation, at least in a temperature range up to 100 degrees C. Also circular dichroism data indicate high resistance of apo A-I to thermal unfolding in the presence of DMPG. It is concluded that the complexes of apo A-I with DMPC are thermodynamically stable only at temperatures near Tc, whereas above and below this temperature range the stability of these recombinants is determined by kinetic factors. In contrast, complexes of apo A-I with DMPG and other acidic phospholipids may be thermodynamically stable over a wide temperature range greater than or equal to Tc. In spite of these fundamental differences between zwitterionic and acidic phospholipids in their mode of association with apo A-I, the binding affinity and the morphology of the recombinants are similar. Both apo A-I X DMPC and apo A-I X DMPG complexes form lipoprotein particles having a discoidal shape.     

Structural studies of apolipoprotein A-I/phosphatidylcholine recombinants by high-field proton NMR, nondenaturing gradient gel electrophoresis, and electron microscopy

Complexes formed between apolipoprotein A-I (apo A-I) and dimyristoylphosphatidylcholine (DMPC) or egg phosphatidylcholine have been studied by high-field 1H NMR, nondenaturing gradient gel electrophoresis, electron microscopy, and gel filtration chromatography. Emphasis has been placed on an analysis of the particle size distribution within the micellar complexes produced at lipid/protein molar ratios of 40-700. As determined by electron microscopy and gel filtration of DMPC/apo A-I complexes, the size of the discoidal micelles produced appears to increase uniformly with an increasing lipid/protein ratio. By electron microscopy, the diameters of isolated DMPC/apo A-I discoidal micelles range from approximately 89 A at a 40 molar ratio to 205 A at a 700 molar ratio. Analysis of the micellar complexes by 1H NMR shows that concomitant with the increase in size is the progressive downfield shift of the choline N-methyl proton resonance of the complex which is observed from 3.245 to 3.267 ppm over the above molar ratio range. The relationship between chemical shift and micelle size is most simply interpreted as arising from a weighted averaging of two lipid environments--lipid-lipid and lipid-protein. In contrast to the above interpretation of the gel filtration experiments on DMPC/apo A-I complexes, nondenaturing gradient gel electrophoresis analysis of particle size distribution leads to an unexpected observation: as the DMPC/apo A-I ratio increases, discrete complexes of increasing size are formed in an apparently quantized manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of lipid exposure of tryptophan residues in membrane peptides and proteins

Fluorescence quenching is used to gain information on the exposure of tryptophan residues to lipid in membrane-bound proteins and peptides. A protocol is developed to calculate this exposure, based on a comparison of quenching efficiency and of a fluorescence lifetime (or quantum yield) measured for a protein and for a model tryptophan-containing compound. Various methods of analysis of depth-dependent quenching are compared and three universal measures of quenching profile are derived. One of the measures, related to the area under profile, is used to estimate quenching efficiency. The method is applied to single tryptophan mutants of a membrane-anchoring nonpolar peptide of cytochrome b(5) and of an outer membrane protein A. Analysis of quenching of the cytochrome's nonpolar peptide by a set of four brominated lipids reveals a temperature-controlled reversible conformational change, resulting in increased exposure of tryptophan to lipid and delocalization of its transverse position. Kinetic quenching profiles and fluorescence binding kinetics reported by Kleinschmidt et al. (Biochemistry (1999) 38, 5006-5016) were analyzed to extract information on the relative exposure of tryptophan residues during folding of an outer membrane protein A. Trp-102, which translocates across the bilayer, was found to be noticeably shielded from the lipid environment throughout the folding event compared to Trp-7, which remains on the cis side. The approach described here provides a new tool for studies of low-resolution structure and conformational transitions in membrane proteins and peptides.     

Intrinsic fluorescence of a hydrophobic myelin protein and some complexes with phospholipids

The fluorescence characteristics of lipophilin, a proteolipid apoprotein from human myelin, were determined in aqueous and lipid environments. In all cases the tryptophan residues were located in buried hydrophobic sites of uniform, but limited, accessibility to the permeant quenching agent acrylamide; only in the helicogenic solvent 2-chloroethanol were the protein fluorophores exposed to the medium. Quantum yields were dependent on the state of aggregation of the protein in aqueous solution and increased considerably on treatment with lysolecithin micelles, or when the protein was combined with phosphatidylcholine by codialysis from 2-chloroethanol into water. Fluorescence titrations indicated that lipophilin bound to lysolecithin with an association constant greater than 10(6) L/mol. Radiationless singlet excitation energy transfer from tyrosine to tryptophan residues was found to decrease markedly when the protein was combined with lipids. When the protein was introduced into dimyristoylphosphatidylcholine vesicles, the tryptophan fluorescence did not detect any solid-liquid phase change. These results were consistent with strong hydrophobic interactions between lipophilin and phospholipids, which lead to conformational adjustments in the protein, and to establishment of an immobilized layer of boundary lipid in bilayer systems.     

Penetration of an emulsion surface by cholesteryl ester transfer protein

Quenching of the intrinsic fluorescence of cholesteryl ester transfer protein (CETP) by spin labelled fatty acids (5-NS and 16-NS) was investigated to determine the degree to which the protein penetrated the phospholipid monolayer surface of a lipid emulsion. When bound to the phospholipid surface approximately 50% of the fluorophores of the transfer protein were accessible to quenching by 5-NS whose nitroxy group locates near the monolayer surface. On the other hand, only 22% of the fluorophores of CETP were accessible to quenching by 16-NS whose nitroxy group locates deeper in the surface monolayer. Quenching of the CETP fluorescence by an aqueous phase quencher (acrylamide) shows that the protein undergoes a conformational change on binding which increases the proportion of the tryptophan residues exposed to the aqueous phase. The results indicate that CETP does not penetrate the lipid surface to a significant degree. Received: 29 March 1996 / Accepted: 30 May 1996     

Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers

Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.     

Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.     

Fluorescence-quenching-resolved spectra of melittin in lipid bilayers

The interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles has been studied by means of fluorescence quenching of the single tryptophan residue of the protein, at lipid-to-peptide ratio, Ri = 50 and at high ionic strength (2 M NaCl). The method of fluorescence-quenching-resolved spectra (FQRS), applied in this study with potassium iodide as a quencher, enabled us to decompose the tryptophan emission spectrum of liposome-bound melittin into components, at temperatures above as well as below the main phase transition temperature (Tt) of DMPC. One of the two resolved spectra exhibits maximum at 342 and 338 nm for experiments above and below Tt, respectively, and is similar to the maximum of tryptophan emission found for tetrameric melittin in solution (340 nm). This spectrum is characterized by the Stern-Volmer quenching constant, Ksv, of about 4 M-1 and it represents the fraction of melittin molecules whose tryptophan residues are exposed to the solvent to a degree comparable with tetrameric species in solution. The other spectrum component, corresponding to the quencher-inaccessible fraction of tryptophan molecules (Ksv = 0 M-1) has its maximum blue-shifted up to 15 nm, indicating a decrease in polarity of the environment. For experiments above Tt, the blue spectrum component revealed the excitation-wavelength dependence, originating probably from the relaxation processes between the excited tryptophan molecules and lipid polar head groups. We conclude that melittin bound to DMPC liposomes exists in two lipid-associated forms; one, with tryptophan residues exposed to the solvent and the other, penetrating the membrane interior, with tryptophan residues located in close proximity to the phospholipid polar head groups of the outer vesicle lipid layer. We also discuss our data with current models of melittin-bilayer interactions.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

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.     

Amphipathic alpha-helix bundle organization of lipid-free chicken apolipoprotein A-I

Apolipoprotein A-I (apoA-I), the major protein component of plasma high-density lipoprotein (HDL), exists in alternate lipid-free and lipid-bound states. Among various species, chicken apoA-I possesses unique structural properties: it is a monomer in the lipid-free state and it is virtually the sole protein component of HDL. Near-UV circular dichroism (CD) spectroscopic studies provide evidence that chicken apoA-I undergoes a major conformational change upon binding to lipid, while far-UV CD data indicate its overall alpha-helix content is maintained during this transition. The fluorescence emission wavelength maximum (excitation 295 nm) of the tryptophans in apoA-I (W74 and W107) displayed a marked blue shift in both the lipid-free (331 nm) and HDL-bound (329 nm) states, compared to free tryptophan in solution. The effect of aqueous quenchers on tryptophan fluorescence was determined in lipid-free, dimyristoylphosphatidylcholine (DMPC)- and HDL-bound states. The most effective quencher in the lipid-free and HDL-bound states was acrylamide, giving rise to Ksv values of 1.6 +/- 0.1 and 1.2 +/- 0.1 M-1, respectively. Together, these data suggest that a hydrophobic environment around the two tryptophan residues (W74 and W107) is maintained in alternate conformations of the protein. To further probe the molecular organization of lipid-free apoA-I, its effect on the fluorescence properties of 8-anilino-1-naphthalenesulfonic acid (ANS) was determined. Human and chicken apoA-I induced a similar increase in ANS fluorescence quantum yield, in keeping with the hypothesis that these proteins adopt a similar global fold in the absence of lipid. When considered with near- and far-UV CD experiments, the data support a model in which lipid-free chicken apoA-I is organized as an amphipathic alpha-helix bundle. In other studies, lipid-soluble quenchers, 5-, 7-, 10-, and 12-DOXYL stearic acid (DSA), were employed to investigate the depth of penetration of apoA-I into the surface monolayer of spherical HDL particles. 5-DSA was the most effective quencher, suggesting that apoA-I tryptophan residues localize near the surface monolayer, providing a structural rationale for the reversibility of apoA-I-lipoprotein particle interactions.     

Properties of lipid-apolipoprotein association products. Complexes of human apo AI and binary phospholipid mixtures

The products resulting from the association of human apo A-I with binary phospholipid mixtures of dimyristoyl phosphatidylcholine (DMPC) and either dipalmitoyl (DPPC) or distearoyl (DSPC) phosphatidylcholine have been isolated and characterized. Effective lipid . protein complex formation was found to occur at the onset temperature for melting of the gel state, and equal incorporation of both lipid components of the binary mixture was observed. Two sizes of products were obtained, one containing 2 A-I molecules per complex and the other containing 3; the proportions of these two products depended upon the initial phospholipid/protein ratio employed. these two product species were found to be resolvable by density gradient centrifugation as well as gel filtration, reflecting substantial differences in composition as well as size. The ratio of DMPC to DPPC or DSPC was the same in the isolated complexes as in the initial mixture, suggesting that th protein does not associate preferentially with the fluid phase lipid, but with lipid domains in which the components are randomly distributed. Electron microscopy of recombinant particles containing a 2:1 ratio (w/w) of DSPC to DMPC revealed stacks of discs whose thickness was proportionately greater than for discs containing DMPC alone. Also of significance was the finding that recombinant discs containing 3 A-I molecules possessed a diameter approximately 1.5 times larger than recombinant discs containing 2 A-I molecules. These data are consistent with the model for the recombinant particles described by Tall et al. (Tall, A.R., Small, D.M., Deckelbau, R.J., and Shipley, G.G. (1977) J. Biol. Chem. 252; 4701-4711), in which the phospholipid is found as a circular bilayer, the thickness of which is dependent upon the length of the acyl chain, and around which the protein is distributed as an annulus.     

The effect of temperature and lipid on the conformational transition of gramicidin A in lipid vesicles

The present study investigated the effect of temperature and lipid/peptide molar ratio on the conformational changes of the membrane peptide gramicidin A from a double-stranded helix to a single-stranded helical dimmer in 1,2-dimyristoyl-glycerol-3-phosphochloine (DMPC) vesicles. Tryptophan fluorescence spectroscopy results suggested that the conformational transition fitted a three-state (two-step) "folding" model. Rate constants, k(1) and k(2), were determined for each of the two steps. Since k(1) and k(2) increased with an increase in temperature, we hypothesized that the process corresponded to the breakage and formation of the backbone hydrogen bonds. The k(1) was from 10 to 45 folds faster than k(2), except for lipid/peptide molar ratios above 89.21, where k(2) increased rapidly. At molar ratios below 89.21, k(2) was insensitive to changes in lipid concentration. To account for this phenomenon, we proposed that while the driving interaction at high molar ratios is between the indole rings of the tryptophan residues and the lipid head groups, at low molar ratios there may be an intermolecular interaction between the tryptophan residues that causes gramicidin A to form an organized aggregated network. This aggregated network, caused by the tryptophan-tryptophan interaction, may be the main effect responsible for the slow down of the conformation change.     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.