Design of a Functional Membrane Protein by Engineering a Heme-Binding Site in Glycophorin A

2007 ◽  
Vol 129 (3) ◽  
pp. 512-518 ◽  
Author(s):  
Jeanine M. Cordova ◽  
Pamela L. Noack ◽  
Simon A. Hilcove ◽  
James D. Lear ◽  
Giovanna Ghirlanda
Keyword(s):  
Membrane Protein ◽  
Binding Site ◽  
Glycophorin A ◽  
Heme Binding ◽  
Functional Membrane

scholarly journals The CytochromecFold Can Be Attained from a Compact Apo State by Occupancy of a Nascent Heme Binding Site

2001 ◽  
Vol 276 (49) ◽  
pp. 45813-45817 ◽  
Author(s):  
Rachel Wain ◽  
Thelma A. Pertinhez ◽  
Esther J. Tomlinson ◽  
Lin Hong ◽  
Christopher M. Dobson ◽  
...  
Keyword(s):  
Binding Site ◽  
Heme Binding ◽  
Apo State

scholarly journals Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr)

2020 ◽  
Vol 295 (32) ◽  
pp. 11316-11325
Author(s):  
Dayeon Nam ◽  
Yuki Matsumoto ◽  
Takeshi Uchida ◽  
Mark R. O'Brian ◽  
Koichiro Ishimori
Keyword(s):  
Binding Site ◽  
Fluorescence Anisotropy ◽  
Response Regulator ◽  
Binding Activity ◽  
Control Element ◽  
Heme Binding ◽  
Cluster Region ◽  
Ice Binding ◽  
Iron Response Regulator ◽  
Ice Complex

The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum. Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr–ICE complex. The addition of EDTA to the Irr–ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr–ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29–mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr–ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29.

scholarly journals Highly malleable haem-binding site of the haemoprotein HasA permits stable accommodation of bulky tetraphenylporphycenes

RSC Advances ◽  
2019 ◽  
Vol 9 (32) ◽  
pp. 18697-18702 ◽  
Author(s):  
Erika Sakakibara ◽  
Yuma Shisaka ◽  
Hiroki Onoda ◽  
Daiki Koga ◽  
Ning Xu ◽  
...  
Keyword(s):  
Binding Site ◽  
Heme Binding ◽  
Flexible Loops

Bulky metallo-tetraphenylporphycene was successfully incorporated into haemophore HasA which have flexible loops surrounding heme-binding site.

scholarly journals Selective detergent-extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes

1998 ◽  
Vol 330 (2) ◽  
pp. 667-674 ◽  
Author(s):  
J. Willem DeGRIP ◽  
Jenny VanOOSTRUM ◽  
Petra H. M. BOVEE-GEURTS
Keyword(s):  
Membrane Protein ◽  
Inclusion Compounds ◽  
Selective Extraction ◽  
Molar Ratio ◽  
Cyclodextrin Complexes ◽  
Protein Ratio ◽  
Cyclodextrin Inclusion ◽  
Discontinuous Sucrose Gradient ◽  
Detergent Extraction ◽  
Functional Membrane

A novel generic approach is described for the selective extraction of detergents from mixed detergent/lipid/protein micelles for the preparation of proteoliposomes of defined lipid-protein ratio. The approach is based on the much higher affinity of inclusion compounds of the cyclodextrin type for detergents in comparison with bilayer-forming lipids. This approach has distinct advantages over other procedures currently in use. It produces good results with all detergents tested, independent of type and critical micelle concentration, and appears to be generally applicable. It yields nearly quantitative recovery of membrane protein in the proteoliposome fraction. Finally, no large excess of lipid is required; a molar ratio of lipid to protein of 100 to 1 already produces proteoliposomes with functional membrane protein, but higher ratios are well tolerated. The size of the vesicles thus obtained depends on the detergent used. Separation of the resulting proteoliposomes from the detergent-cyclodextrin complexes was most easily achieved by centrifugation through a discontinuous sucrose gradient. A variety of detergents was tested in this procedure on the bovine rod visual pigment rhodopsin in combination with retina lipids. In all cases good yields of proteoliposomes were obtained, which contained fully functional rhodopsin.

Oxygen diffusion near the heme binding site of horseradish peroxidase

1991 ◽  
Vol 178 (1) ◽  
pp. 104-109 ◽  
Author(s):  
Victor Vargas ◽  
Juan E. Brunet ◽  
David M. Jameson
Keyword(s):  
Horseradish Peroxidase ◽  
Binding Site ◽  
Oxygen Diffusion ◽  
Heme Binding
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