1M6T

CRYSTAL STRUCTURE OF B562RIL, A REDESIGNED FOUR HELIX BUNDLE

1M6T の概要

• エントリーDOI: 10.2210/pdb1m6t/pdb
• 関連するPDBエントリー: 256B
• 分子名称: Soluble cytochrome b562, SULFATE ION (3 entities in total)
• 機能のキーワード: protein design, electron transport
• 由来する生物種: Escherichia coli
• 細胞内の位置: Periplasm: P0ABE7
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 11881.29

構造登録者

Chu, R.,Takei, J.,Knowlton, J.R.,Andrykovitch, M.,Pei, W.,Kajava, A.V.,Steinbach, P.J.,Ji, X.,Bai, Y. (登録日: 2002-07-17, 公開日: 2002-11-06, 最終更新日: 2023-08-30)

主引用文献

Chu, R.,Takei, J.,Knowlton, J.R.,Andrykovitch, M.,Pei, W.,Kajava, A.V.,Steinbach, P.J.,Ji, X.,Bai, Y.
Redesign of a Four-Helix Bundle Protein by Phage Display Coupled with Proteolysis and Structural Characterization by NMR and X-ray Crystallography
J.Mol.Biol., 323:253-262, 2002

PubMed Abstract: To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.

PubMed: 12381319
DOI: 10.1016/S0022-2836(02)00884-7

実験手法

X-RAY DIFFRACTION (1.81 Å)