Paolo Cavatorta

Fluorescence depolarization in liquid crystals and membrane bilayers

Abstract The paper introduces in a systematic way the concepts of order parameters and correlation functions needed for a description of orientational ordering in a liquid crystal or a membrane bilayer. The strong collision and the diffusion model for reorientation are examined in some detail. After discussing the main characteristics of the very popular probes 1,6 diphenylhexatriene (DPH) and perylene the theory of rotational depolarization of fluorescence for a probe in an ordered phase is introduced. Time-dependent and steady state experiments in monodomains are discussed together with their angular dependence. Attention is then focussed on membrane vesicles. The theory for fluorescence depolarization is presented for probes with any orientation of the transition moments with a view to investigating the feasibility of using a certain probe to extract information on ordering and dynamics. Applications of the fluorescence depolarization technique to model and biological membranes are briefly reviewed. An appendix introducing irreducible tensors and Wigner rotation matrices properties is provided in order to keep the paper reasonably self-contained.

Conformation of bovine myelin basic protein purified with bound lipids.

Abstract 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 α-helix and β-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.

Lipid peroxidation and fluidity of plasma membranes from rat liver and Morris hepatoma 3924A

Plasma membranes isolated from the fast‐growing, maximal‐deviation, Morris hepatoma 3924A exhibit remarkable changes in lipid composition, lipid peroxidation and to some extent in the physical state with respect to rat liver plasmalemmas. A correlation appears to exit between the lower phospholipid: protein ratio, higher cholesterol: phospholipid ratio, lower rate of lipid peroxidation and decrease in fluidity in tumor plasma membranes.

EXCITED STATE pKa BEHAVIOUR OF DAPI. A RATIONALIZATION OF THE FLUORESCENCE ENHANCEMENT OF DAPI IN DAPI‐NUCLEIC ACID COMPLEXES

Abstract— The steady state and time resolved fluorescence of the drug and chromosomal staining agent, 4′,6‐diamidino‐2‐phenylindole dihydrochloride, DAPI, was examined under different solvent conditions. In solutions between pH 3 and pH 9 the fluorescence spectral maximum of DAPI was found at 460 nm. The fluorescence decayed with double exponential kinetics, with decay times of 2.86 and 0.144 ns, at all wavelengths below 550 nm. At 550 nm single exponential decay kinetics with a lifetime of 0.153 ns was observed. The fluorescence spectrum could be resolved into two components, the 2.86 ns component having a spectral maximum near 450 nm and the 0.144 ns component having a spectral maximum near 490 nm. The results are rationalized in terms of there being two different configurations of DAPI, one of which undergoes a rapid protonation of the indole ring by proton transfer from the 6‐amidinium group in the excited singlet state. The 0.144 ns component is assigned as the fluorescence from the excited state of the protonated indole ring. The results provide an explanation of the fluorescence enhancement in DAPI‐nucleic acid complexes.

Specificity of zinc binding to myelin basic protein

Z2+ 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 of65Zn 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.

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.

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.

On the interactions of metal ions with tryptophan and glyciltryptophan: a fluorescence study

Abstract The interaction of copper (Cu2+) and nickel (Ni2+) with tryptophan (Trp) and glyciltryptophan (GlyTrp) was studied by monitoring the quenching of fluorescence due to complex formation. pH titration of mixtures of M2+/L show that Cu2+ lowers the pKa for the NH+3 group from 9.36 to 5.9 in Trp and from 8.2 to 5.5 in Glytrp while the effect of Ni2+ is relatively smaller. Association constants were evaluated from Stern-Volmer plots and are shown to be 1.9×105 M-5 for Cu2+-Trp, 4.4×104 M-1 for Cu2+-GlyTrp, 4.0×104 M-1 for Ni2+-Trp and 6.9×103 M-1 for Ni2+-GlyTrp measured at pH 9. The binding of metals to Trp or GlyTrp at pH 7 leads to very different constants for Ni2+ and Cu2+, due to enhanced complex formation between the aniomic form of the compounds and the metal ions. The binding of nickel at this pH is strongly reduced.

Tryptophan interactions of gramicidin A′ channels in lipids: a time-resolved fluorescence study

The evolution of the incorporation of cation transport channels into lysolecithin micelles by gramicidin A was followed by measuring the ns time-resolved fluorescence of the tryptophan residues. In all samples, the tryptophan fluorescence could be resolved into three exponentially decaying components. The three decay times ranged from 6 to 8 ns, 1.8 to 3 ns, and 0.3 to 0.8 ns, depending on the emission wavelength. The fractional fluorescence of each component changed with incubation time. The long lifetime component had a reduced contribution to the total fluorescence while the short decay time component increased. The fluorescence spectra could be resolved into three distinct fluorescent components having maxima at 340 nm, 330 nm and 323 nm after 90 min of incubation, and 335 nm, 325 nm and 320 nm after 24 h of incubation. These maxima were, respectively, associated with the long, medium and short decay components. The fluorescence decay behaviour was interpreted as representing three families of tryptophans, the short lifetime component being due to a stacking interaction between tryptophan residues. The variation with incubation time suggests a two-step process in the channel-lipid organization. The first is associated with the conformational change of the polypeptide as it takes up a left-handed helical head-to-head dimer structure in the lipid. The second step is proposed to involve changes originating from membrane assembly and intermolecular interactions between channels as they form hexameric clusters.

Interaction of copper and nickel ions with tryptophan and glyciltryptophan: Time resolved flourescence study

Abstract The interaction of copper (Cu++) and nickel (Ni++) with tryptophan (Trp) and glyciltryptophan (GlyTrp) was studied monitoring the quenching of flourescence due to complex formation. The deviations from linear Stern-Volmer plots for both metals were treated in a different way reflecting the difference in binding. In the case of nickel the Lehrer formalism was used which permitted us to estimate the binding constant as well as the fraction of fluorophore accessible to the metal. In the case of copper the Hill plot formalism was more appropriate giving a degree of cooperativity upon binding as well as the association constant of the complex. The values of the constants are in agreement with our previous values based on Stern-Volmer plots. Time resolved fluorescence measurement were also performed to assess the mechanism of quenching as well as the contribution from different conformers in solution. The fluoresscence decay is best described as a triple exponential with lifetimes of 7.9, 2.7 and 0.23 ns at 25°C. The quenching is essentially static, the lifetimes remain constant and changes are observed only in the pre-exponentials. In the case of nickel the shortest lifetime remains essentially unquenched while the other two components are effectively quenched. In the case of copper the quenching depends on the metal concentration but all of the conformers are quenched significantly. Our results suggest the assignment of one of the conformers as being the short lifetime component in the decay of fluorescence.