Streptavidin binding to biotinylated lipid layers on solid supports. A neutron reflection and surface plasmon optical study

Abstract

Neutron reflection and surface plasmon optical experiments have been performed to evaluate structural data of the interfacial binding reaction between the protein streptavidin and a solid-supported lipid monolayer partly functionalized by biotin moieties. Since both experimental techniques operate in a total internal reflection geometry at a substrate/solution interface, identical sample architectures allow for a direct comparison between the results obtained with these two recently developed methods. It is found that a monomolecular layer of dipalmitoyllecithin doped with 5 mol% of a biotinylated-phosphatidylethanolamine shows a thickness of d1 approximately (3.4 +/- 0.5) nm. Binding of streptavidin to the biotin groups results in an overall layer thickness of d = (5.9 + 0.5) nm that demonstrates the formation of a well-ordered protein monolayer with the (biotin+spacer) units of the functionalized lipids being fully embedded into the binding pocket of the proteins. It is demonstrated by model calculations that a more detailed picture of the internal structure of this supramolecular assembly can only be obtained if one uses deuterated lipid molecules, thus generating a high contrast between individual layers.

Interfacial assembly of proteins and peptides: recent examples studied by neutron reflection

“…The biomolecular systems that have so far been studied include proteins (Lu et al ,d, 1999a(Lu et al ,b,c, 2003bSu et al 1998aSu et al ,b,c, 1999Su et al , 2000Fragneto et al 2000b;Holt et al 2000;Nylander et al 2001;Armstrong et al, 2004;Cooper et al 2005;Xu et al 2006bXu et al , 2007Xu et al , 2008Pan et al 2008), peptides (Holt et al 2000;Lu et al 2003aLu et al , 2004Lad et al 2007;Middelberg et al 2008;Zhao et al 2009), biosurfactants (Follows et al 2007) and lipid bilayers (Fragneto et al 2000a. A number of neutron reflection studies have also focused on interfacial interactions from binary mixtures of protein/surfactant (Lu et al 1998a,b;Green et al 2000aGreen et al ,b, 2001, protein or peptide/lipid (Schmidt et al 1992;Lu et al 2001;Miano et al 2007;Valincius et al 2008), protein/polymer Hollmann et al 2007Hollmann et al , 2008; Ganzevles et al 2008), DNA/polymer (Zhao et al 2008a), DNA/surfactant (Zhang et al 2008) and DNA/ lipid (Wu et al 2006). Several recent reviews have been dedicated to covering most of these systems (Lu 1999(Lu , 2002Fragneto-Cusani 2001;Krueger 2001;Thomas 2004;…”

Small Noncytotoxic Carbon Nano‐Onions: First Covalent Functionalization with Biomolecules

“…The values for the thicknesses of the layers presented in this paper are also in agreement with values already published in the literature. [29,48,49] The structures containing CNOs were also examined by the SPR method. The different procedures of ox-CNO incorporation into SAMs are described in Scheme 1.…”

Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes

“…calculated with constant refractive index, the "adlayer thickness" becomes a measure of molecule density; only at close packing it can be interpreted in terms of molecular dimensions. According to published data obtained with well-defined model systems, n = 1.45 is a reasonable value for the refractive index of lipids in a fluid state and adsorbed proteins (Lang et al, 1992(Lang et al, , 1994Spinke et al, 1992Spinke et al, , 1993Reiter et al, 1993; Terrettaz et ai., 1993) (and references therein).5 For lipids and fatty acid salts in a crystalline state, refractive indices in the range of n = 1.5 seem more appropriate (Swalen, 1986;Kooyman & Krull, 1991;Schmidt et al, 1992). But in our experiments, a crystalline state of lipids on the waveguide can be excluded (see below).…”

“…This difference cannot be accounted for by the slightly different masses of POPC and biotin-DPPE. Literature data on thickness of supported DMPC and DPPC monolayers doped with 5 mol% biotin-DPPE range from 16 A to 31 A (Schmidt et al, 1992) at n = 1.50 (corresponding to 23 A or 44 A , respectively, evaluated at n = 1.45).…”