1kl3
From Proteopedia
|
an engineered streptavidin with improved affinity for the strep-tag II peptide : SAm1-StrepII
Overview
The Strep-tag II is a nine-amino acid peptide that was developed as an, affinity tool for the purification of corresponding fusion proteins on, streptavidin columns. The peptide recognizes the same pocket of, streptavidin where the natural ligand is normally bound so that biotin or, its chemical derivatives can be used for competitive elution. We report, here the crystal structures of the streptavidin mutants '1' and '2,' which, had been engineered for 10-fold higher affinity towards the Strep-tag II., Both streptavidin mutants carry mutations at positions 44, 45, and 47, that is, in a flexible loop region close to the binding site. The crystal, structures of the two apo-proteins and their complexes with the Strep-tag, II peptide were refined at resolutions below 2 A. Both in the presence and, absence of the peptide, the lid-like loop next to the ligand, pocket--comprising residues 45 through 52--adopts an 'open' conformation, in all four subunits within the asymmetric unit. The same loop was, previously described to be disordered in the wild-type apo-streptavidin, and to close over the pocket upon complexation of the natural ligand, biotin. Our findings suggest that stabilization of the 'open' loop, conformation in the absence of a ligand abolishes the need for, conformational rearrangement prior to the docking of the voluminous, peptide. Because no direct contacts between the flexible part of the loop, and the peptide ligand were detected, it seems likely that the higher, affinity of the two streptavidin mutants for the Strep-tag II is caused by, a predominantly entropic mechanism.
About this Structure
1KL3 is a Single protein structure of sequence from Streptomyces avidinii. Full crystallographic information is available from OCA.
Reference
Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site., Korndorfer IP, Skerra A, Protein Sci. 2002 Apr;11(4):883-93. PMID:11910031
Page seeded by OCA on Tue Nov 20 19:20:06 2007
