title: Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin.
abstract: 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.
authors.affiliation: Department of Biological Structure, University of Washington, Box 357420, Seattle, WA 98195-7420, USA.
authors.forename: Isolde
authors.initials: I
authors.lastname: Le Trong
authors.forename: Vano
authors.initials: V
authors.lastname: Chu
authors.forename: Yi
authors.initials: Y
authors.lastname: Xing
authors.forename: Terry P
authors.initials: TP
authors.lastname: Lybrand
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Stenkamp
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; C066114:biotin-streptavidin complex; D001710:Biotin; D019809:Streptavidin
keywords: biotin; biotin-binding protein; circular permutation