scholarly journals Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies

2013 ◽  
Vol 10 (79) ◽  
pp. 20120717 ◽  
Author(s):  
Daniel A. Andrews ◽  
Meng Xie ◽  
Victoria Hughes ◽  
Matthew C. Wilce ◽  
Anna Roujeinikova
Keyword(s):  
Membrane Proteins ◽  
Leucine Zipper ◽  
Integral Membrane Protein ◽  
Flagellar Motor ◽  
Structural Studies ◽  
Transmembrane Helices ◽  
Biophysical Analysis ◽  
Torque Generation ◽  
Low Levels

The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This ‘solubilization by design’ approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.

scholarly journals Detergent-free extraction, reconstitution and characterization of membrane-anchored cytochrome-b5 in native lipids

2020 ◽  
Vol 56 (48) ◽  
pp. 6511-6514 ◽  
Author(s):  
Bankala Krishnarjuna ◽  
Thirupathi Ravula ◽  
Ayyalusamy Ramamoorthy
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Cytochrome B5 ◽  
Target Protein ◽  
Structural Studies ◽  
Net Charge

Directly extracted membrane proteins are stable and suitable for functional and high-resolution structural studies; however, the efficacy is modulated by the net charge of the target protein and the polymer used at a given pH.

scholarly journals Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation

2008 ◽  
Vol 190 (20) ◽  
pp. 6660-6667 ◽  
Author(s):  
Yong-Suk Che ◽  
Shuichi Nakamura ◽  
Seiji Kojima ◽  
Nobunori Kami-ike ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
High Speed ◽  
Proton Translocation ◽  
Energy Coupling ◽  
Flagellar Motor ◽  
Transmembrane Helices ◽  
Wild Type ◽  
Torque Generation ◽  
Serovar Typhimurium ◽  
Bacterial Flagellar Motor ◽  
Protein Instability

ABSTRACT MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.

scholarly journals Mutants of theYarrowia lipolytica PEX23Gene Encoding an Integral Peroxisomal Membrane Peroxin Mislocalize Matrix Proteins and Accumulate Vesicles Containing Peroxisomal Matrix and Membrane Proteins

2000 ◽  
Vol 11 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Trevor W. Brown ◽  
Vladimir I. Titorenko ◽  
Richard A. Rachubinski
Keyword(s):  
Membrane Proteins ◽  
Sequence Similarity ◽  
Integral Membrane Protein ◽  
Functional Complementation ◽  
Matrix Proteins ◽  
Hypothetical Proteins ◽  
Peroxisomal Membrane ◽  
High Sequence Similarity ◽  
Low Levels ◽  
Yeast Yarrowia Lipolytica

pex mutants are defective in peroxisome assembly. The mutant strain pex23-1 of the yeast Yarrowia lipolytica lacks morphologically recognizable peroxisomes and mislocalizes all peroxisomal matrix proteins investigated preferentially to the cytosol. pex23 strains accumulate vesicular structures containing both peroxisomal matrix and membrane proteins. The PEX23 gene was isolated by functional complementation of the pex23-1 strain and encodes a protein, Pex23p, of 418 amino acids (47,588 Da). Pex23p exhibits high sequence similarity to two hypothetical proteins of the yeastSaccharomyces cerevisiae. Pex23p is an integral membrane protein of peroxisomes that is completely, or nearly completely, sequestered from the cytosol. Pex23p is detected at low levels in cells grown in medium containing glucose, and its levels are significantly increased by growth in medium containing oleic acid, the metabolism of which requires intact peroxisomes.

3D Crystallization and Structural Studies of Integral Membrane Proteins in Lipidic Cubic phases

2000 ◽  
Vol 56 (s1) ◽  
pp. s83-s83
Author(s):  
P. Nollert ◽  
M. L. Chiu ◽  
M. C. Loewen ◽  
A. Royant ◽  
H. Behrhali ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Structural Studies ◽  
Cubic Phases

scholarly journals Mating Type in Chlamydomonas Is Specified by mid, the Minus-Dominance Gene

Genetics ◽  
1997 ◽  
Vol 146 (3) ◽  
pp. 859-869 ◽  
Author(s):  
Patrick J Ferris ◽  
Ursula W Goodenough
Keyword(s):  
Transcription Factor ◽  
Mating Type ◽  
Codon Bias ◽  
Leucine Zipper ◽  
Laboratory Strain ◽  
Leucine Zipper Motif ◽  
Type Locus ◽  
Diploid Cells ◽  
Necessary And Sufficient

Diploid cells of Chlamydomonas reinhardtii that are heterozygous at the mating-type locus (mt  +/mt  –) differentiate as minus gametes, a phenomenon known as minus dominance. We report the cloning and characterization of a gene that is necessary and sufficient to exert this minus dominance over the plus differentiation program. The gene, called mid, is located in the rearranged (R) domain of the mt  – locus, and has duplicated and transposed to an autosome in a laboratory strain. The imp11 mt  – mutant, which differentiates as a fusion-incompetent plus gamete, carries a point mutation in mid. Like the fus1 gene in the mt  + locus, mid displays low codon bias compared with other nuclear genes. The mid sequence carries a putative leucine zipper motif, suggesting that it functions as a transcription factor to switch on the minus program and switch off the plus program of gametic differentiation. This is the first sex-determination gene to be characterized in a green organism.

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