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

Schultz-Heienbrok, R. and Maier, Timm and Strater, N.. (2004) Trapping a 96 degrees domain rotation in two distinct conformations by engineered disulfide bridges. Protein Science, 13 (7). pp. 1811-1822.

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Official URL: http://edoc.unibas.ch/45828/

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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 12degrees 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 considerable residual domain flexibility. In the double mutant that was designed to trap the enzyme in the closed conformation, the structure analysis reveals an unexpected intermediate conformation along the 96degrees rotation trajectory between the open and closed enzyme forms. A comparison of the five independent conformers analyzed in this study shows that the domain movement of the variant enzymes is characterized by a sliding movement of the residues of the domain interface along the interface, which is in contrast to a classical closure motion where the residues of the domain interface move perpendicular to the interface.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Maier)
UniBasel Contributors:Maier, Timm
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Cambridge University Press
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:03 Nov 2017 11:23
Deposited On:03 Nov 2017 11:23

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