Previous analyses of limited proteolytic sites within native, folded protein structures have shown that a significant conformational change is required in order to facilitate binding into the active site of the attacking proteinase. For the serine proteinases, the optimum conformation to match the proteinase binding-site geometry has been well characterized crystallographically by the conserved main-chain geometry of the reactive site loops of their protein inhibitors. A good substrate must adopt a conformation very similar to this "target" main-chain conformation prior to cleavage. Using a "loop-closure" modeling approach, we have tested the ability of a set of tryptic-limited proteolytic sites to achieve this target conformation and further tested their suitability for cleavage. The results show that in most cases, significant changes in the conformation of at least 12 residues are required. All the putative tryptic cleavage sites in 1 protein, elastase, were also modeled and tested to compare the results to the actual nicksite in that protein. These results strongly suggest that large local motions proximate to the scissile bond are required for proteolysis, and it is this ability to unfold locally without perturbing the overall protein conformation that is the prime determinant for limited proteolysis.