PDBsum entry 1m6t

Electron transport

PDB id: 1m6t

Contents

Protein chain

106 a.a. *

Ligands

SO4

Waters ×118

* Residue conservation analysis

PDB id:

1m6t

Name:

Electron transport

Title:

Crystal structure of b562ril, a redesigned four helix bundle

Structure:

Soluble cytochrome b562. Chain: a. Synonym: cytochrome b562. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: cytb562. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.81Å R-factor: 0.210 R-free: 0.248

Authors:

R.Chu,J.Takei,J.R.Knowlton,M.Andrykovitch,W.Pei,A.V.Kajava, P.J.Steinbach,X.Ji,Y.Bai

Key ref:

R.Chu et al. (2002). 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. PubMed id: 12381319 DOI: 10.1016/S0022-2836(02)00884-7

Date:

17-Jul-02 Release date: 06-Nov-02

Protein chain

P0ABE7  (C562_ECOLX) -  Soluble cytochrome b562 from Escherichia coli

Seq: 128 a.a.

Struc: 106 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DOI no: 10.1016/S0022-2836(02)00884-7

J Mol Biol 323:253-262 (2002)

PubMed id: 12381319

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography.

R.Chu, J.Takei, J.R.Knowlton, M.Andrykovitch, W.Pei, A.V.Kajava, P.J.Steinbach, X.Ji, Y.Bai.

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.

Selected figure(s)

Figure 2.
Figure 2. Ribbon diagrams of (a) apocyt. b[562][24.] and (b) cyt. b[562][43.] with helix I in red, helices II and III in cyan, and helix IV in green. The heme and selected side-chains are shown as ball-and-stick. Carbon atoms are colored gray, nitrogen atoms blue, oxygen atoms red, sulfur atoms yellow, and the iron orange. It should be noted that the unfolded region of apocyt. b[562] may not be as fully unfolded as shown in (a). Some residual structures may exist.[44.] Prepared with the programs MOLSCRIPT [45.] and Raster3D. [46.]

Figure 7.
Figure 7. Three-dimensional structures of cyt. b[562],[41.] RIL (this work) and ING (this work). (a) Superposition of the crystal structure of RIL (green ribbon) and cyt. b[562] (cyan ribbon and ball-and-stick heme). (b) Averaged NMR solution structure of ING. (c) Crystal structure of RIL. Prepared with the programs MolScript[45.] and Raster3D. [46.]

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 323, 253-262) copyright 2002.

Figures were selected by an automated process.