Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin

Circular permutation of streptavidin was carried out in order to investigate the role of a main‐chain amide in stabilizing the high‐affinity complex of the protein and biotin. Mutant proteins CP49/48 and CP50/49 were constructed to place new N‐termini at residues 49 and 50 in a flexible loop involved in stabilizing the biotin complex. Crystal structures of the two mutants show that half of each loop closes over the binding site, as observed in wild‐type streptavidin, while the other half adopts the open conformation found in the unliganded state. The structures are consistent with kinetic and thermodynamic data and indicate that the loop plays a role in enthalpic stabilization of the bound state via the Asn49 amide–biotin hydrogen bond. In wild‐type streptavidin, the entropic penalties of immobilizing a flexible portion of the protein to enhance binding are kept to a manageable level by using a contiguous loop of medium length (six residues) which is already constrained by its anchorage to strands of the β‐barrel protein. A molecular‐dynamics simulation for CP50/49 shows that cleavage of the binding loop results in increased structural fluctuations for Ser45 and that these fluctuations destabilize the streptavidin–biotin complex.