X‐ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome <i>b</i><sub>5</sub> Phe35→Tyr mutant

Yao, Ping; Wu, Jian; Wang, Yunhua; Sun, Bingyun; Xia, Zong-Xiang; Huang, Zhong‐Xian

doi:10.1046/j.1432-1033.2002.03120.x

Cited by 12 publications

(6 citation statements)

References 60 publications

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“…WT-Tb5 and its other mutants yielded good crystals which diffracted to 1.8±2.1 A Ê reso- lution and the re®ned structures gave R factors of around 19% (Xue et al, 1999;Wu et al, 2000Wu et al, , 2002Gan et al, 2002;Wang, Wu et al, 2002;Yao et al, 2002), including the F58Y mutant presented in this paper. However, it was exceedingly dif®cult to grow single crystals of the F58W mutant.…”

Section: Discussionsupporting

confidence: 50%

“…5(b) and 5(c). In WT-Tb5 one of the propionic acids of the heme is hydrogen bonded to Ser64 (Wu et al, 2000;Durley & Mathews, 1996) and its conformation is conserved in all the mutants of Tb5 (Xue et al, 1999;Gan et al, 2002;Wu et al, 2002;Wang, Wu et al, 2002;Yao et al, 2002), including F58Y; however, the other propionic acid group is extended into the solvent and is more¯exible. The extended groups also exhibit multiple conformations in the F58W mutant (Figs.…”

Section: F58y F58wmentioning

confidence: 99%

“…Cytochrome b 5 is an electron-transfer protein that is widely distributed in nature. The structure±function relationship of cytochrome b 5 has been studied based on the crystal structures of mutants of cytochrome b 5 , the microsomal cytochrome b 5 and the outer mitochondrial membrane cytochrome b 5 (Funk et al, 1990;Rivera et al, 1998;Xue et al, 1999;Wu et al, 2000Wu et al, , 2002Gan et al, 2002;Wang, Wu et al, 2002;Yao et al, 2002;Cowley et al, 2002;Ren et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Structure of the F58W mutant of cytochromeb₅: the mutation leads to multiple conformations and weakens stacking interactions

Gan

et al. 2005

Acta Crystallogr D Biol Cryst

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Phe58 of cytochrome b5 is involved in stacking interactions with heme and the axial ligand His63. To elucidate the contribution of the stacking interactions to protein stability, the crystal structures of the F58Y and F58W mutants were determined at high resolution. The structure of the F58Y mutant is basically the same as that of the wild-type protein. However, the mutation from Phe58 to Trp58 leads to difficulty in growing single crystals and results in a space-group change; the six molecules in the asymmetric unit form two groups that are related by a non-crystallographic twofold axis. The structure of F58W was determined using molecular replacement by making use of the non-crystallographic symmetry. The F58W mutation gives rise to multiple conformations of six side chains, a peptide linking two of the six residues and the extended propionic acid of the heme. The six molecules in the asymmetric unit of the F58W mutant structure are grouped into two types based on their conformations and one of the six molecules exhibits dual conformations. The stacking interactions are weakened owing to the increase/decrease of the angles between the indole ring of Trp58 and the His63/heme rings, which accounts for the lower stability of F58W compared with the wild-type protein.

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Section: Discussionsupporting

confidence: 50%

Section: F58y F58wmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structure of the F58W mutant of cytochromeb₅: the mutation leads to multiple conformations and weakens stacking interactions

Gan

et al. 2005

Acta Crystallogr D Biol Cryst

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“…Stability is also decreased by the mutation of Phe63 involved in a -stacking interaction with the axial ligand His68 (Wang et al, 2006). The redox potential is decreased by the mutation of Phe40 located near the haem plane (Yao et al, 2002) and by the mutation of Ser69 interacting with the haem propionate group (Funk et al, 1990). The mutation of Val50 and Val66 also influences the redox potential of the haem (Gan et al, 2002;Wu et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

High-resolution crystal structures of the solubilized domain of porcine cytochromeb₅

Hirano

Kimura

Tamada

2015

Acta Cryst D Biol Crystallogr

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“…More recently, recombinant forms truncated to only the heme-binding cytb5 soluble domain (cytb5-sd) have been extensively used for biophysical analysis and structural determinations [2,18]. The codon-optimized rat cytb5-sd [19] and the tobacco cytb5 [20] have been expressed in E. coli.…”

Section: Introductionmentioning

confidence: 99%

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

Sobrado

Goren

James

et al. 2008

Protein Expression and Purification

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A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of ~18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3 mg per]of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.

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X‐ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b₅ Phe35→Tyr mutant

Cited by 12 publications

References 60 publications

Structure of the F58W mutant of cytochromeb₅: the mutation leads to multiple conformations and weakens stacking interactions

Structure of the F58W mutant of cytochromeb₅: the mutation leads to multiple conformations and weakens stacking interactions

High-resolution crystal structures of the solubilized domain of porcine cytochromeb₅

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

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X‐ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35→Tyr mutant

Cited by 12 publications

References 60 publications

Structure of the F58W mutant of cytochromeb5: the mutation leads to multiple conformations and weakens stacking interactions

Structure of the F58W mutant of cytochromeb5: the mutation leads to multiple conformations and weakens stacking interactions

High-resolution crystal structures of the solubilized domain of porcine cytochromeb5

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

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Product

Resources

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X‐ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b₅ Phe35→Tyr mutant

Structure of the F58W mutant of cytochromeb₅: the mutation leads to multiple conformations and weakens stacking interactions

Structure of the F58W mutant of cytochromeb₅: the mutation leads to multiple conformations and weakens stacking interactions

High-resolution crystal structures of the solubilized domain of porcine cytochromeb₅