Laminar order within Langmuir-Blodgett multilayers from phospholipid and myelin basic protein: a neutron reflectivity study.

Multilayers consisting of negatively charged phospholipid DMPA and myelin basic protein (MBP) were assembled by Langmuir-Blodgett deposition of floating Langmuir monolayers from the air/water interface to solid substrates. Protein/lipid samples were obtained by binding MBP from the aqueous subphase to the phospholipid monolayers before deposition. The vertical organization of these model membranes (i.e., with organization perpendicular to the substrate surface) was investigated in detail by neutron reflectivity measurements, and the internal distribution of water molecules was determined from the change of contrast after in-situ H2O/D2O exchange. The multilayers were well ordered, with repeating lipid bilayers as fundamental structural unit. MBP was inserted in between adjacent lipid headgroups, such as in the natural myelin membrane. Water molecules in the multilayers were present mainly in the lipid headgroup and protein slab. On exposition of the pure lipid multilayers to a dry atmosphere, a reduction of the bilayer spacing was determined, whereas the global lamellar order was not affected. In contrast, drying of the protein/lipid multilayers induced degradation of the laminar order. The data demonstrate that ordered Langmuir-Blodgett multilayers are versatile model systems for studying how competing interactions between lipid, protein, water, and ions affect the global organization of such multilamellar lipid/protein assemblies. Here, the water molecules were found to be a necessary mediator to maintain the laminar order in a multilayer from DMPA and myelin basic protein.

Small angle x-ray scattering from lipid-bound myelin basic protein in solution.

The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH < 3.0 from defatted brain, is the classical preparation of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extracted from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution.

An x-ray absorption spectroscopy study of the zinc environment in Langmuir-Blodgett phospholipid multilayers.

For the first time x-ray absorption spectroscopy was used to investigate the Zn environment in Langmuir-Blodgett multilayers. The multilayers were taken as a model of the multilamellar structure of the myelin sheath, the membrane surrounding the nerve axon, which plays a crucial role for signal transduction along the axon. The layers were assembled from the phospholipid dilauroylphosphatidic acid, both in the presence and in the absence of myelin basic protein. The analysis of the extended x-ray absorption fine structure and of the near edge regions of the x-ray absorption spectra at the Zn K-edge provided an accurate description of the local structure showing that the Zn ions are bound to the heads of the phospholipid molecules. The myelin basic protein induces a distortion on the Zn local environment due to a steric constraint but does not substitute the phosphate headgroups. These findings represent an important step in understanding the interplay among myelin basic protein, Zn, and the lipids of the myelin sheath.

Determining the binding capability of the mouse major urinary proteins using 2-naphthol as a fluorescent probe.

The mouse major urinary proteins (MUPs) are an ensemble of isoforms secreted by adult male mice and involved in sexual olfactory communication. MUPs belong to the lipocalin superfamily, whose conserved structure is a beta-barrel made of eight antiparallel beta-strands forming a hydrophobic pocket that accommodates small organic molecules. A detailed knowledge of the molecular mechanism associated to the binding of those molecules can guide protein engineering to devise mutated proteins where the ligand specificity, binding affinity, and release rate can be modulated. Proteins with such peculiar properties may have interesting biotechnological applications for pest control, as well as in food and cosmetic industries. In this work, we demonstrate that the fluorescent molecule 2-naphthol binds to the natural ligand's binding site of MUPs with high affinity. In addition, we show that 2-naphthol binds to MUPs in its protonated form, that its fluorescence is blue-shifted, and the quantum yield is increased, thus confirming the high hydrophobicity of the protein pocket and the absence of proton acceptors inside the binding site. At large the results presented, besides demonstrating that the use of 2-naphthol provides a convenient and quick method for testing MUPs binding activity and to ascertain the quality of the protein preparation, suggest that MUPs can represent an interesting system for studying the photophysical characteristics of fluorescent molecules in a highly hydrophobic environment.

Kinetic and structural study of the interaction of myelin basic protein with dipalmitoylphosphatidylglycerol layers.

The interaction of myelin basic protein (MBP) with dipalmitoylphosphatidylglycerol films has been investigated by means of a microgravimetric gauge sensitive to the changes in load and structural modifications of the layer deposited onto its surface. Fourier transform infrared spectroscopy, circular dichroism, and x-ray diffraction have confirmed protein uptake by the lipid phase along with a global disordering effect onto the lipid alkyl chains and have shown a temporal evolution of the structure of water penetrating the lipid phase together with the protein. These effects are clearly related to the temporal variation of the microgravimetric gauge signal. Finally, measurements carried out on pre-annealed samples point out the role of mesoscopic morphology in determining the pathways through which MBP penetrates the lipid multilayer. The results obtained in our model system could be useful in clarifying the mechanisms of the myelinating and demyelinating processes that take place in the natural membrane.

Conformation of bovine myelin basic protein purified with bound lipids.

The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having alpha-helix and beta-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results.

Purification of bovine P2 myelin protein with bound lipids.

The P2 protein is a neuritogenic, small basic protein present in PNS myelin. It belongs to the family of the cytoplasmic lipid-binding proteins and can be incorporated in lipidic bilayers. P2 has been purified and crystallized only in the lipid-free form. Here we show that the P2 protein can be purified with bound lipids by applying to PNS myelin the same procedure that as used to purify lipid-bound myelin basic protein from CNS myelin. SDS-PAGE showed a single band of 16.5 kDa, and TLC showed the presence of most of the myelin lipids associated with the protein. Lipid-bound P2 revealed different circular dichroism spectra from the corresponding lipid-free form, indicating that lipids influence P2 conformation.

Specificity of zinc binding to myelin basic protein.

Zn2+ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of 65Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.

An oxygenation-sensitive dye binding to Carcinus maenas hemocyanin.

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.

Myelin basic protein interaction with zinc and phosphate: fluorescence studies on the water-soluble form of the protein.

The interaction of myelin basic protein (MBP) with zinc and phosphate ions has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out by means of both static and time-resolved fluorescence techniques. The addition of either zinc to MBP in the presence of phosphate or phosphate to MBP in the presence of zinc resulted in an increase of fluorescence intensity and a blue shift of the emission maximum wavelength. Furthermore, a concomitant increase in the scattering was also detected. Anisotropy decay experiments demonstrated that these effects are due to the formation of MBP molecules into large aggregates. A possible physiological role for such interaction is discussed.

The interaction of DAPI with phospholipid vesicles and micelles.

The interactions of the dye 4',6-diamidino-2-phenylindole (DAPI) with phospholipids ordered in single bilayer vesicles of dioleylphosphatidylserine (DOPS) or dimyristoylphosphatidylcholine (DMPC) or micelles of monomyristoylphosphatidylcholine (MPC) have been investigated. Somewhat unexpectedly, the binding of this dye to such ordered structures is not affected by the ionic strength of the external medium, which suggests an embedding of DAPI into the hydrocarbon phase. The fluorescence enhancement of DAPI bound can be accomodated within a model previously proposed for the behaviour of DAPI bound to proteins (Mazzini et al., Biophys. Chem. 42 (1992) 101). From both static and dynamic anisotropy measurements, bound DAPI results severely restricted in its rotational freedom but insensitive to the temperature dependent phase transition of the saturated DMPC vesicles. The considerable tightness and specificity of the interactions between DAPI and ordered phospholipids are also deduced from preliminary fluorescence quenching studies (reduced accessibility of iodide ions towards DAPI bound and quenching effects by the chaotrop Nonidet P-40).

Fluorescence studies on the conformation of litorin in solution and in the presence of model membranes.

The conformation of the nonapeptide hormone litorin has been studied in buffer and in the presence of lipids, using static and dynamic fluorescence. The results obtained show that, in buffer, the hormone probably exists in a collection of flexible conformers, slowly interconverting between them. The marked changes observed in fluorescence spectra and lifetimes upon addition of dimyristoylphosphatidylserine vesicles clearly show that the peptide interacts with lipids assuming lipid specific conformations. Interestingly, no significative spectroscopic changes are produced by exposure to dimirystoylphosphatidylcholine vesicles both in the gel and liquid-christalline phases, suggesting a requirement for negatively charged lipids during the process of hormone-membrane interaction.

The interaction of 4', 6-diamidino-2-phenylindole (DAPI) with pepsin.

We recently found that the fluorescent dye DAPI, well-known for its use with nucleic acids, is also able to interact with proteins as well as ordered phospholipids assemblies. The interaction of DAPI with pepsin under different conditions of pH and ionic strength was studied with fluorescence and circular dichroism techniques. From a comparison of the results obtained, the interaction appears to be rather tight and specific, dependent on both electrostatic and hydrophobic forces, and able to probe the tridimensional conformation of the protein.

The binding of 4',6-diamidino-2-phenylindole to bovine serum albumin.

The binding of 4',6-diamidino-2-phenylindole (DAPI) to bovine serum albumin (BSA) has been investigated between pH 6 and 8, in 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations and the results analyzed according to a procedure previously reported (R. Favilla and A. Mazzini, Biochim. Biophys. Acta 788 (1984) 48). The dye binds to the protein with a blue shift of about 4 nm in its fluorescence emission maximum, but with an enhancement factor of 10 of its fluorescence quantum yield. The dissociation constant decreases from 100 microM to 54 microM as the pH is increased from 6 to 8, with a constant number of nearly three equivalent binding sites. The complete displacement of DAPI bound to BSA by Ca2+ suggests a possible specificity of this substantially electrostatic interaction. The fluorescence decay of DAPI bound to the protein shows a double exponential kinetics, with a tau 1 = 0.97 ns and tau 2 = 2.78 ns. These results, compared with those obtained for DAPI alone, tau 1 = 0.16 ns and tau 2 = 2.8 ns, are rationalized in terms of two different rotamers of DAPI. Both rotamers are able to bind to the protein, but only one of them undergoes an intramolecular proton transfer, from the 6-amidinium group to the indole aromatic ring, in the excited singlet state of DAPI alone. When DAPI interacts with BSA this transfer does not occur and consequently a large increase of fluorescence is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence and CD studies on the conformation of the gastrin releasing peptide in solution and in the presence of model membranes.

The conformation of the heptacosapeptide hormone, gastrin releasing peptide, has been studied in buffer and in the presence of lipids, using static and dynamic fluorescence and CD. The results obtained show that, in buffer, the hormone exists in a collection of flexible, random coil type conformers, characterized by a beta-turn between residues 14-19. On the other hand, organic solvents can induce some degree of ordered secondary structure in the peptide chain. The marked changes, observed in CD and fluorescence spectra upon addition of lysolecitin micelles and dimyristoylphosphatidylserine vesicles, clearly show that the peptide interacts with lipids, assuming a lipid specific configuration. Interestingly, no significative spectroscopic changes are produced by exposure to dimyristoylphosphatidylcholine vesicles both in the gel and liquid-chrystalline phases, suggesting a requirement for negatively charged lipids during the process of hormone-membrane interaction.

Interaction of cations with lipid-free myelin basic protein. A spectroscopy study.

The interaction of some divalent cations with myelin basic protein (MBP) in buffer and in model membranes was studied by using the static fluorescence of the intrinsic tryptophan residue of the protein. Results were indicative of Zn++ ability to bind to MBP. The observed binding could facilitate the interaction of MBP with lipids and have a role in stabilizing the myelin sheath.

Postnatal maturation of rat intestinal membrane: lipid composition and fluidity.

1. We studied the lipid composition and the fluidity of small intestine brush border membrane (BBM) of rats of different age: 'very young' (5-7 weeks old), 'young' (9 weeks old), 'adult' (30 weeks old) and 'old' (85 weeks old). 2. Fluorescence anisotropy, as assessed by 1,6-diphenyl-1,3,5-hexatriene probe (DPH), was increased from very young to adult rats. 3. In agreement with these results the lipid composition in adult animals showed a lower lipid/protein ratio (derived mainly from a lower content of total polar lipids) and an increase of cholesterol esters and sphingomyelin (SM) saturation index. 4. A marked decrease of the order parameter was observed in the 'old' group, accompanied by a decreased cholesterol/phospholipid ratio. 5. The percentage distribution of membrane phospholipids significantly changed during development, but the modifications were not correlated with the anisotropy of DPH.

Estimation of protein secondary structure from circular dichroism spectra: a critical examination of the CONTIN program.

The computer program CONTIN uses the Provencher and Glöckner procedure to calculate protein secondary structure from circular dichroism spectra. We have tested this program with peptides and proteins in which unfolding was either induced by denaturing treatment or was already present. Results indicate that the program does not clearly discriminate between the ordered and the unordered states of a protein.

Lipid peroxidation and physical properties of smooth and rough hepatoma microsomes.

Smooth and rough endoplasmic reticulum of two Morris hepatomas, the slow growing 9618A and the fast growing 3924A, have been isolated, and their biochemical composition, supramolecular organization, and response to the action of peroxidative agents have been studied. Cytochrome P450 content and lipid availability are the limiting factors of their peroxidizability. The hemoprotein content is reduced about 80% in hepatoma 9618A and is virtually absent in hepatoma 3924A. The peroxidizability decreases with increasing growth rate of the tumor. The protein, phospholipid, and cholesterol content, the fatty acid composition as well as the double bond index, and the saturated and unsaturated fatty acid content are reported. Differences have been found between normal liver and tumors and between the fractions within a given tumoral tissue. The molecular order, as determined by fluorescence anisotrophy decay of DPH, increases in total microsomes and in the smooth fraction going from liver 9618A to 3924A, whereas for the rough fraction it is the same in liver and hepatoma 9618A; in 3924A it increases of about 30%. Fluidity decreases in total microsomes going from liver to 3924A, to 9618A. In both the purified fractions it decreases with increasing deviation of the tumor.

Conformation of bombesin in buffer and in the presence of lysolecithin micelles: NMR, CD, and fluorescence studies.

The conformation of the tetradecapeptide hormone bombesin has been studied in buffer and in the presence of lysolecithin micelles, using static and dynamic fluorescence, CD, and one- and two-dimensional nmr. The results obtained show that in buffer bombesin is present in an extended flexible chain, with no evidence for any ordered secondary structure. A marked change in the CD spectrum is observed changing from buffer to the lipid suspension. Concomitantly, the 1H-nmr spectrum of bombesin, in a D2O lipid dispersion, shows the persistence of resonances due to exchangeable protons and in similar conditions the fluorescence intensity increases. We think therefore that these results strongly support the hypothesis that bombesin interacts with the lipid phase, assuming ordered secondary structure. Finally, the marked dependence of tryptophan fluorescence quantum efficiency and order parameter from the hormone concentration in the presence of lysolecithin but not in buffer leads to the conclusion that bombesin can associate into the lipid matrix.