Direct force measurements, surface plasmon resonance spectroscopy, and genetic manipulations were used to investigate the impact of the orientation of immobilized cytochrome b5 (cyt b5) on its interactions with cytochrome c (cyt c). In this work, we used two cyt b5 orientational variants that present different regions of the protein surface when immobilized. Direct force measurements demonstrated that the two orientations generate a small difference in the electrostatic surface potential of the protein monolayers, in agreement with the calculated electrostatic potential distribution across the protein surface. This difference did not result in any differences in the electrostatic force between cyt c and the cyt b5 variants, however. The measured equilibrium binding constant for the cyt c interaction with cyt b5 also did not depend on the orientation of the latter. These results suggest that, at large separations, cyt c initially interacts relatively nonspecifically with a large patch of negative charge on the cyt b5. At short separations, it then adopts the optimum relative orientation for electron transfer. The force measurements not only elucidated the molecular basis of the equilibrium binding behavior, but also the possible molecular mechanisms that govern the interactions between these proteins in solution.

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.