scholarly journals The structure of hydrogenase-2 from Escherichia coli: implications for H2-driven proton pumping

2018 ◽  
Vol 475 (7) ◽  
pp. 1353-1370 ◽  
Author(s):  
Stephen E. Beaton ◽  
Rhiannon M. Evans ◽  
Alexander J. Finney ◽  
Ciaran M. Lamont ◽  
Fraser A. Armstrong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quaternary Structure ◽  
Anaerobic Respiration ◽  
Integral Membrane Protein ◽  
Proton Pumping ◽  
Membrane Bound ◽  
Hydrogenase 2 ◽  
Overexpression System ◽  
H2 Oxidation

Under anaerobic conditions, Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from hydrogen (H2) oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe–S protein (HybA) and an integral membrane protein (HybB). To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of Hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In the present paper, we describe a new overexpression system that has facilitated the determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex.

Membrane-bound carbonic anhydrase in the heart

1992 ◽  
Vol 262 (2) ◽  
pp. H577-H584 ◽  
Author(s):  
W. Bruns ◽  
G. Gros
Keyword(s):  
Carbonic Anhydrase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Integral Membrane Protein ◽  
Rabbit Heart ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
The One ◽  
Triton X

Microsomal membranes from bovine heart homogenates were subfractionated by density gradient centrifugation. Fractions with high levels of a sarcolemmal (SL) marker are enriched in specific carbonic anhydrase (CA) activity up to ninefold compared with the microsomes. Fractions with high levels of a sarcoplasmic reticulum marker and a mitochondrial marker, respectively, exhibit specific CA activities that are similar to the one found in the microsomes. Determination of cytosolic markers reveals that the CA activity in the SL fraction is not due to contamination by cytosolic CA, and it is shown by Triton X-114 phase separation that the CA activity is due to an integral membrane protein. In cryosections from rabbit heart the SL region of cardiomyocytes is stained by the fluorescent CA inhibitor dansylsulfonamide. Intracellular staining occurs also, with a pattern suggesting the presence of CA associated with intracellular membranes. Although it cannot be excluded that there is a contribution by endothelial membranes, it appears likely that most CA of the heart is bound to the SL. The possible involvement of the enzyme in extracellular proton buffering is discussed.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

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