Recognition processes at a functionalized lipid surface observed with molecular resolution

Abstract

The specific binding of proteins to functionalized lipid monolayers on aqueous subphases was characterized by neutron reflectivity and fluorescence microscopy measurements. Due to the high affinity and high specificity of their noncovalent interaction, streptavidin (SA) and biotin (vitamin H) were chosen as a model system to investigate the structural characteristics of a recognition process on a molecular length scale. Changes in the neutron reflection from the surfaces of NaCl aqueous (H2O or D2O) protein solutions (10(-8) M SA) were used to monitor the interaction of the protein with a monolayer of a biotinylated lipid in situ. Refinement of the reflectivity data and independent fluorescence microscopic observation of the interface using FITC-labeled SA showed that the protein forms macroscopically homogeneous (and presumably crystalline) domains covering a large portion of the surface. Moreover, the neutron reflection experiments clearly showed the formation of a monomolecular protein layer with an effective thickness, dp = 43.7 +/- 2 A. The area per protein molecule occupied in the film was A0 = 2860 +/- 200 A2 and nw = 260 +/- 100 water molecules were associated with each protein molecule. Quantitative binding was found to occur at biotin surface concentrations as low as 1 molecule/1,250 A2 (compared with approximately 1 molecule/40 A2 for dense packing). This study demonstrates the application of a promising new tool for the systematic investigation of molecular recognition processes in protein/lipid model systems.

Direct molecular level measurements of the electrostatic properties of a protein surface

“…After draining the apparatus, the organic surface layers, including the protein monolayers and the lipid bilayers, were burnt away by UV irradiation in an atmosphere of filtered N 2 . The measured thickness of 172 Ϯ 3 Å for two supported protein monolayers is consistent with the protein and bilayer thicknesses determined by x-ray diffraction, force measurements, and neutron reflectivity (22,29,36).…”

“…The fractional protein surface coverage of 85 Ϯ 3% was determined both by quantifying the amount of 125 I radiolabeled protein bound to the bilayers (27) and by surface plasmon resonance spectroscopy (28). The measured coverage agreed with results from neutron reflectivity studies of crystalline streptavidin domains on floating biotin-lipid films (29).…”

The Analysis and Interpretation of Neutron and X-ray Specular Reflection

“…Of special interest is the possibility of including X-ray reflectivity data in the fitting scheme. Several authors have used parallel fitting of models to the neutron and X-ray profiles from the same system to determine the interfacial structure (Styrkas, Thomas, Adib, Davis, Hodge & Liu, 1994;Vaknin, Als-Nielsen, Peipenstock & Losche, 1991) but do not seem to have used a single minimizing routine. The larger ~ range of the X-ray profile presents no difficulty when the reflectivity is expressed in terms of partial structure factors.…”

Ion distributions at charged aqueous surfaces: Synchrotron X-ray scattering studies

“…Volume, in-plane density of electrons, etc., are among such constraints that can be used. Applying such constraints [76,77,78] can reduce the uncertainties and make ED profiles associated to the actual molecular arrangement. During the fitting process (minimizing the χ 2 ), χ 2 could be easily trapped into a serial local minima, making the initial estimating values important to reach the global minimum.…”