Molecular Level Characterization of Microenvironmental Influences on the Properties of Immobilized Proteins

Abstract

This work examines the molecular influence of the local environment on the apparent properties of immobilized proteins. Using the surface force apparatus and cytochrome b5 immobilized to different supports, we measured directly the range and magnitude of molecular forces originating from the underlying matrix. In particular, we used as supports oriented streptavidin monolayers, charged maleimide-functionalized phospholipid bilayers, and neutral maleimide-functionalized lipid bilayers. The relative impact of the underlying surface forces on the apparent electrostatic properties of the bound proteins was evaluated, as was the efficacy of different methods such as electrostatic screening and charge neutralization on the ability to minimize their influence. These background forces not only mask structural features of the proteins that may direct ligand trajectories to their binding sites, but they also alter the efficiencies of protein coupling to the solid support. The results presented provide valuable information for the design of immobilization matrices.

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

“…Soluble streptavidin binds to these layers and self-assembles into twodimensional crystals (26). 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).…”

“…2). We thereby determined the effective charge projected into a plane tangent to the protein surface (20,27). With identical samples, the measured surface charge density was independent of the salt concentration (data not shown).…”

“…1 shows the distance dependence of the electrostatic double-layer force between two streptavidin monolayers at pH 6.0. Fits of the long-range, repulsive force to the one-dimensional nonlinear Poisson-Boltzmann equation gave the electrostatic surface charge density and surface potential (22,23,27). The best fits were obtained with the constant charge boundary condition (31).…”

“…In measurements that used a second charged bilayer, the test membrane contained 85 mol % 1,2 ditridecanoyl-sn-glycero-3-phosphocoline and 15 mol % of the negatively charged, synthetic lipid N-(4-maleimidomethylcyclohexyl-L-carbonyl)1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine. The surface charge density of the latter monolayer is Ϫ11 mC͞m 2 (27).…”

Direct Evidence That Neural Cell Adhesion Molecule (NCAM) Polysialylation Increases Intermembrane Repulsion and Abrogates Adhesion

“…Quantification of Protein Surface Densities-To determine the surface density of proteins immobilized on the NTA-TRIG-DLGE supported bilayers, we measured the amount of bound 125 I-labeled protein, according to procedures described previously (22,23).…”