Trapping a 96 degrees domain rotation in two distinct conformations by engineered disulfide bridges.

ABSTRACT: Engineering disulfide bridges is a common technique to lock a protein movement in a defined conformational state. We have designed two double mutants of Escherichia coli 5'-nucleotidase to trap the enzyme in both an open (S228C, P513C) and a closed (P90C, L424C) conformation by the formation of disulfide bridges. The mutant proteins have been expressed, purified, and crystallized, to structurally characterize the designed variants. The S228C, P513C is a double mutant crystallized in two different crystal forms with three independent conformers, which differ from each other by a rotation of up to 12 degrees of the C-terminal domain with respect to the N-terminal domain. This finding, as well as an analysis of the domain motion in the crystal, indicates that the enzyme still exhibits conside

SUBMITTER: Schultz-Heienbrok R 

PROVIDER: S-EPMC2279917 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature