Penetration and Saturation of Lysozyme in Phospholipid Bilayers

Abstract

We show that the combination of X-ray reflectivity, tryptophan fluorescence spectrum, and fluorescence quenching by bromine provides a useful tool to probe the location of lysozyme in lipid bilayers. To this end, we prepare lamellar complexes composed of phospholipids and lysozyme on solid surfaces using a solutioncasting method. The proteins lie spontaneously between adjacent bilayers in the complexes. The results indicate that lysozyme may penetrate into the lipid bilayers. But the penetration depth is very shallow, and the tryptophan residues do not penetrate beyond the interface between the hydrocardon core and the headgroup region of the lipid bilayer. The penetration becomes saturated when more proteins are incorporated into the lamellar complex. The excess proteins stay in the interlamellar aqueous layers.

Susceptibility of lysozyme to in-vitro digestion and immunoreactivity of its digests

“…environment of the protein making it more hydrophobic, possibly through membrane binding. In fact, a blue shift in the spectrum of LYS has been attributed to a shielding effect from water arising from the penetration of the protein into a phospholipid bilayer (Yuan et al, 2007). When different amounts of PC were titrated into a fixed concentration of LYS at acidic pH the fluorescence intensity decreased regularly (Fig.…”

“…The biological functions of LYS, such as its antimicrobial and immunomodulating properties, have been attributed to its ability to interact with membrane component phospholipids and to penetrate into lipid bilayers, aspects which have been broadly studied (Gorbenko, Loffe, & Kinnunen, 2007;Yuan et al, 2007). Similarly, the interaction of LA with phospholipid membranes and vesicles is of interest because membranes are implicated in the protein folding behaviour and in the ability of LA to regulate lactose synthesis (Cawthern et al, 1996).…”

Electrostatically driven lipid–protein interaction: Answers from FRET

“…Nevertheless, the thickness of the interbilayer aqueous space, δ w = 2 × h~5-6 nm, recovered in this study is now closer to the value measured earlier by performing FRET measurements between two membrane probes (D = BODIPY-PC; A = Rh-PE) in the presence of lysozyme (δ w = 6.7 nm [87]). This interbilayer thickness is wide enough to accommodate monomeric, or even oligomeric, sideways-on or headways-on membrane-bound lysozyme molecules, particularly if lysozyme partially penetrates the membrane surface at a high protein surface coverage [89]. Altogether, these complementary FRET results, performed both at the bulk (liposome ensemble-average) and microscopic (single-fiber) levels, provided important topological information about the supramolecular organization of the lipid-lysozyme mixed fibers, confirming that they display a multilayer structure, in which the predominantly hexameric lysozyme is sandwiched between two adjacent lipid bilayers.…”

FRET studies of lipid‐protein aggregates related to amyloid‐like fibers

“…For higher protein concentrations (≥ 2.0 μM in the studied system) bridging at the pinches is so widespread (because of the higher protein surface concentration) that even the interbilayer distance sensed by FRET from membrane probe donors is now somewhat reduced (because of obvious restriction in the extent of membrane bending between neighboring pinches), though not nearly as much as that reported by FRET from protein donors. Additionally, for these higher concentrations, it is expected that lysozyme inserts into the bilayer to some extent (Yuan et al. 2007; Gorbenko et al.…”

“…For higher protein concentrations ( ‡ 2.0 lM in the studied system) bridging at the pinches is so widespread (because of the higher protein surface concentration) that even the interbilayer distance sensed by FRET from membrane probe donors is now somewhat reduced (because of obvious restriction in the extent of membrane bending between neighboring pinches), though not nearly as much as that reported by FRET from protein donors. Additionally, for these higher concentrations, it is expected that lysozyme inserts into the bilayer to some extent (Yuan et al 2007;Gorbenko et al 2008), reducing the lamellar spacing even further. It is hypothesized that the decrease in the dielectric constant experienced locally by lysozyme at a high protein/ lipid ratio upon extensive protein-induced liposome aggregation might be responsible for the loosening of its tertiary structure, ultimately causing its disruption and formation of amyloid-like fibers.…”