Spectroscopic analysis of acylated and deacylated myelin proteolipid protein

Abstract

The effect of covalently bound fatty acid on the conformation of the myelin proteolipid protein has been studied by ultraviolet and intrinsic fluorescence spectroscopy. With dimethyl sulfoxide used as a perturbant, the exposure of Trp and Tyr residues in various mixtures of chloroform-methanol was evaluated by difference spectroscopy of the proteolipid protein (APL) and its chemically deacylated form (d-APL). The fraction of chromophoric groups exposed increased with the proportion of chloroform with 25% of the groups exposed in 1:2 chloroform-methanol and 98% in 3:1 chloroform-methanol. These conformational changes correlate well with changes in intrinsic viscosity. Values for the deacylated form were indistinguishable from those of the acylated protein, suggesting that fatty acids do not affect protein conformation in organic solvents. In water, UV difference spectroscopy indicated that the number of Tyr and Trp groups exposed in both APL and d-APL was relatively small and was independent of the molecular size of the perturbant. However, differences in the environment of the Trp groups in the two forms of the protein could be demonstrated by intrinsic fluorescence. When the protein was excited at 295 nm, the maximum emission wavelength for the acylated protein was 330 nm, whereas it was 335 nm for the deacylated form. Furthermore, the Trp groups in d-APL were more easily quenched by acrylamide than in APL, indicating that they were more exposed, or in a more hydrophilic environment, following deacylation. Protein aggregation appears to be independent of the presence of fatty acids, suggesting that the fluorescence differences between APL and d-APL are related to factors other than aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical deacylation reduces the adhesive properties of proteolipid protein and leads to decompaction of the myelin sheath

“…We have previously shown that incubation of isolated PLP with neutral HA results in the quantitative deacylation of the protein (Bizzozero and Lees 1986; Bizzozero and Good 1990). However, prior to assessing the effect of deacylation on myelin structure, it was necessary to determine if treatment of nervous tissue with HA also removes the PLP‐bound fatty acids.…”

“…Appropriate blanks consisting of vesicles without protein were prepared and their absorbances at 450 nm were subtracted out from the sample values. The amount of protein incorporated into vesicles was assessed by the high‐speed centrifugation technique (Bizzozero and Lees 1986). In some experiments, PLP‐induced vesicle aggregation was monitored by light microscopy using a Olympus BX60 Microscope.…”

“…1989) clearly indicate that some of the protein functions are mediated or regulated by covalently bound fatty acids. In the case of PLP, our laboratory has previously shown that the removal of fatty acid by treatment with neutral hydroxylamine induces large conformational changes (Bizzozero and Lees 1986). Therefore, it is reasonable to speculate that fatty acids may play a role on the only known function of the protein (i.e.…”

The Light-induced Reactions of Tryptophan with Halocompounds¶

“…The quenching of Trp fluorescence in proteins by chloroform has been used to study the location of Trp residues in proteins (8), but to interpret the results of chloroformquenching experiments, it is necessary to appreciate the importance of the photochemical reaction (5). Chloroform has also been used as a solvent for the solubilization of a variety of integral membrane proteins, such as the c-subunit of ATP synthase (9)(10)(11), myelin proteins (12,13) and small multidrug-resistant proteins (14,15). The findings presented here demonstrate that UV illumination of the protein in chloroform-containing solvents may lead to photodestruction of the Trp residues.…”

Effect of ATP Depletion on the Palmitoylation of Myelin Proteolipid Protein in Young and Adult Rats

“…In cell‐free systems, PLP is palmitoylated with acyl‐CoA by a nonenzymatic mechanism (Bizzozero et al, 1987), and it is depalmitoylated by a specific fatty acylesterase (Bizzozero et al, 1992). Although the physiological role of the thioesterification cycles and the specific function of the acyl moieties are presently unknown, the removal of fatty acids produces large conformational changes that are thought to affect the biological activity of the protein (Bizzozero and Lees, 1986 a ).…”