The molecular size and shape of the Folch-Pi apoprotein in aqueous and organic solvents.

In 1% acetic acid, sedimentation velocity measurements and equilibrium ultracentrifuge experiments demonstrate that the Folch-Pi apoprotein is not monodisperse. The weight-average molecular weight calculated from ultracentrifuge experiments and combining sedimentation coefficient and viscosity measurements, ranged from 64000 to 80000. The intrinsic viscosity value suggests an asymetric shape for the apoprotein if a low value of hydration is considered. In dioxan/1% acetic acid (2:3, v/v) a smaller sedimentation coefficient was found, the intrinsic viscosity value remaining identical to that in 4% acetic acid. In pure 2-chloroethanol, light-scattering experiments led to a molecular weight of 165000 indicating that even in this solvent the protein is not monomeric. Intrinsic viscosity and light scattering measurements on the one hand, primary sequence on the other hand (six proline residues per monomer of Mr 23500) suggest that the molecule in 2-chloroethanol may consist of rod-like segments with flexible junctions.     

A study of Folch-Pi apoprotein. II. Relation between polymerization state and conformation.

A comparison of the conformation of Folch-Pi apoprotein in organic solvent and in aqueous solutions has been made by ESR, infrared and circular dichroism spectroscopy studies. Electrophoresis and ultracentrifugation have been carried out in order to correlate molecular weight and charge of the molecule with its conformation. It appears that the protein is monomeric in organic solution. In water, only one component is present but the molecules behave as a polydisperse system of associating molecules. Hydrophobic interacitons seem to be important for this polymerisation which does not appear to be accompanied by the formation of beta-structure. After the transfer of the protein from organic solution to water, the ESR spectra of the protein labelled on the free SH groups show an heterogeneity in the motional environment of the label which permits to assume that different areas of association exist in the polymeric molecule.     

Study of fluorescent tryptophyl residues and extrinsic probes for the characterization of molecular domains of Folch-Pi apoprotein.

The highly hydrophobic myelin Folch-Pi apoprotein can be solubilized in organic as well as in aqueous media. In order to understand the molecular organization changes consecutive to changes in the solvent medium, the environment of intrinsic probes and extrinsic labels has been studied by fluorescence and accessibility to some reagents. In acqueous solution, only two tryptophan residues per protein molecule of 23,500 molecular weight have been shown to fluoresce, and their fluorescence characterisitics indicate an hydrophobic and/or constrained environment. Two ANS binding sites have also been observed having a high quenching effect on the intrinsic chromophore fluorescence. A large accessibility has been evidenced for the protein sulfhydryl groups in chloroform-methanol 2:1 (v/v), both by kinetic study of the protein reaction with a specific reagent, N-(1-anilino-naphtyl-4) maleimide, and by the fluorescence characteristics of this probe once linked to the protein. The free sulfhydryl groups were still reactive in acqueous solution, but extrinsic fluorescence of the labelled apoprotein transferred from chloroform-methanol 2:1 (v/v) into water gave evidence of constraints on the probe or on its environment. Such constraints may contribute to the solubilization in acqueous solution of this highly hydrophobic protein.     

The thiol groups of the Folch-Pi protein from bovine white matter. Exposure, reactivity and significance.

The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2'-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.     

Myelin Proteins in Reverse Micelles: Tight Lipid Association Required for Insertion of the Folch-Pi Proteolipid into a Membrane-Mimetic System

The solubility and reactivity of the Folch-Pi proteolipid from bovine CNS have been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate, isooctane, and water. Such a membrane-mimetic system resembles the aqueous spaces of the native myelin sheath in terms of its physicochemical properties. Although the proteolipid is completely insoluble in water, it can be inserted into the water-containing micellar system. In contrast, the lipid-depleted protein failed to be incorporated into these organized assemblies. The lipid requirements for insertion of the proteolipid were studied, therefore, after delipidation by several precipitations with isooctane, a nondenaturing solvent. Novel extraction procedures and quantitative analyses by HPLC of the protein-bound lipids revealed the persistence of a lipid-protein complex (6 +/- 1 mol of lipid/mol of protein) displaying optimal micellar solubilization. Competition experiments carried out with brain lipids provide evidence for a preference of the myelin protein for sulfatide, phosphatidylinositol, and phosphatidylserine, in that order. The resulting proteolipid, although differing in relative composition, showed good solubility in the membrane-mimetic system. In contrast, reconstitution experiments carried out with the lipid-depleted protein resulted in weak lipid binding and poor micellar incorporation. These results suggest that the tightly bound acidic lipids may stabilize a protein conformation required for insertion into the micellar system.     

Study of the apoprotein of Folch-Pi bovine proteolipid. II. Characterization of the components isolated from sodium dodecyl sulfate solutions

Acrylamide gel electrophoresis in dodecyl sulfate solutions of Folch-Pi apoprotein shows several bands. The different components were separated by Biogel P-200 filtration and then reduced and carboxymethylated. A comparative study of the amino acid composition, N-terminal sequence and C-terminal amino acid of the different components led to the assumption that their primary sequences are similar. Evidence for a contamination of the protein by free amino acids might explain the difference in terminal groups found by us and by other groups.It has been shown that the purified components can polymerize independently of SS bond formation or exchange. The polymerization products were found to resist dissociation by dodecyl sulfate. It has been suggested therefore that the differences in migration rates of the various components are related to their shape rather than to their molecular weight.     

Study of the apoprotein of Folch-Pi bovine proteolipid. II. Characterization of the components isolated from sodium dodecyl sulfate solutions.

Acrylamide gel electrophoresis in dodecyl sulfate solutions of Folch-Pi apoprotein shows several bands. The different components were separated by Biogel P-200 filtration and then reduced and carboxymethylated. A comparative study of the amino acid composition, N-terminal sequence and C-terminal amino acid of the different components led to the assumption that their primary sequences are similar. Evidence for a contamination of the protein by free amino acids might explain the difference in terminal groups found by us and by other groups. It has been shown that the purified components can polymerize independently of S-S bond formation or exchange. The polymerization products were found to resist dissociation by dodecyl sulfate. It has been suggested therefore that the differences in migration rates of the various components are related to their shape rather than to their molecular weight.     

Structural studies of the apoprotein of the Folch-Pi bovine brain myelin proteolipid : characterization of the CNBr-fragments and of a long C-terminal sequence.

The action of cyanogen bromide on the quite insoluble bovine proteolipid apoprotein allowed the determination of four peptide fragments: two of them constituted a 19 amino acid long C-terminal sequence of the apoprotein. Our results were in favour of the existence of only one subunit presenting a molecular weight closely related to 25,000 for which a schematic representation is given.