Effects of Immobilization Matrices and Protein Orientation on Biomolecular Recognition at Solid-Liquid Interfaces

149 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.The specific recognition between biological molecules has been widely exploited in many applications in medicine, biology, and biotechnology. These applications range from biosensors to affinity chromatography. However, the immobilization of biomolecules, such as proteins, often results in the loss of their biological activities. Factors that impact the apparent kinetics and affinities of the biomolecular interactions at the solid-liquid interfaces were examined with a combination of several physical techniques. The recognition between cytochrome b5 and cytochrome c was chosen as the model system to both identify and quantify the forces that originated from the immobilization substrates and the protein immobilization orientations. These measured forces were correlated with the measured kinetics and affinities of the recognition of immobilized proteins by their binding partners.In this study, the cytochrome b5 mutant T8C was immobilized on different substrates through site-selective immobilization. The range and magnitudes of the force fields due to the immobilization matrices were directly measured using the surface force apparatus (SFA). The apparent electrostatic surface properties of the oriented cytochrome b5 films, as well as the efficacy of the protein coupling to the solid surfaces depend strongly on the forces from the immobilization substrates. These substrate force fields also impact the apparent kinetics and the affinities of selective binding of cytochrome c by soluble cytochrome b5. The latter was measured by the surface plasmon resonance (SPR) spectroscopy. Moreover, the measured magnitudes and ranges of the substrate forces were in good qualitative agreement with the measured surface binding behavior of soluble proteins.We then investigated the dependence of protein orientation on the forces between immobilized proteins and soluble ligands. Oriented cytochrome b5 monolayers were constructed on supported lipid membranes using a site-selective immobilization method. The orientation of the immobilized cytochrome b5 was well controlled as verified with linear dichroism measurements. The orientational dependence of the protein electrostatic surface potentials was observed by direct force measurement. The impact of immobilized cytochrome b5 orientation on the forces mediating the cytochrome b5-cytochrome c recognition was quantified, and correlated with the measured surface binding behaviors. We found good agreement between force measurements, molecular models, and equilibrium binding behavior.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD