The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide

The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for study of recombinant proteins, from detection purification to functional immobilization. To further improve binding Strep-tag streptavidin we have subjected two protruding loops that shape its pocket – instead D-biotin recognized by natural protein iterative random mutagenesis. Sequence analyses hits screening assays revealed several unexpected structural motifs, such a disulfide bridge at base one loop, replacement crucial residue Trp120 Gly two-residue deletion in second loop. mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced towards II, which was boosted case bivalent Twin-Strep-tag®. Four representative mutants were crystallized complex with their X-ray structures solved high resolutions. In addition, crystal structure between XT Twin-Strep-tag elucidated, indicating mode explaining experimentally observed avidity effect. Our illustrates plasticity scaffold reveals interaction modes would been difficult predict. As result, convenient reagent kinetically stable immobilization proteins fused Twin-Strep-tag. possibility reversibly dissociating complexes simply competing enables studies science well cell biology.