We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined.electric double layer ͉ electrostatic simulations ͉ orientation flexibility A dsorption of proteins on solid interfaces has become an area of great theoretical and practical interest because of the recently extensive use of immobilized proteins and applications involving the catalytic potential of immobilized enzymes. Thus with the advent of protein microarrays and other solid miniaturized devices involving proteins, combined with applications in biomedical material engineering and biosensors, a greater need for defining the interactions of proteins with various surfaces has become evident. Accordingly, information on the orientation by which a protein may encounter the surface for immobilization has become an essential requirement. With such information, it would be possible to make predictions regarding details of interactions involving the participating components on the biomolecule and the reactive surface. Moreover, the residues on the biomolecule available for interaction with other proteins and ligands in solution and for binding to the reactive groups at the surface could be identified. Furthermore, customized surface chemistries resulting in a desirable immobilization orientation would become facilitated, resulting in a more targeted approach to protein immobilization.Despite the increasing need to understand how proteins attach to cellular and artificial surfaces, experimental details of the orientation by which proteins are immobilized are, to our knowledge, available only in two cases [type I hexokinase and mitochondrial creatine kinase (MtCK)] because of the difficulty in obtaining reliable experimental data. As for membrane proteins, the structure of only a limited number of cases (e.g., bacteriorhodopsin, and microsomal P450, see refs. 1 and 2) have been determined, because of the inherent difficulties associated with their crystallization. The other main stru...