Structural comparison of substrate entry gate for rhomboid intramembrane peptidasesThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 216-223 ◽  
Author(s):  
Christelle Lazareno-Saez ◽  
Cory L. Brooks ◽  
M. Joanne Lemieux
Keyword(s):  
Membrane Proteins ◽  
Hydrophobic Interactions ◽  
Peer Review Process ◽  
Mobile Element ◽  
Transmembrane Helices ◽  
General Role ◽  
E Coli ◽  
Signalling Molecules ◽  
Health And Disease ◽  
Selection Of

Rhomboids are intramembrane serine peptidases conserved in all kingdoms of life. Their general role is to cleave integral membrane proteins to release signalling molecules. These signals, when disrupted, can contribute to various diseases. Crystal structures of H. influenzae (hiGlpG) and E. coli GlpG (ecGlpG) rhomboids have revealed a structure with six transmembrane helices and a Ser–His catalytic dyad buried within the membrane. One emerging issue was the identification of the mobile element in the protein that allows substrate docking. It has been proposed that the substrate entry gate is composed of helix 5 and loop 5. The present review studies the structures of these two orthologs. In ecGlpG structures, different conformations of loop 5 and helix 5 are observed. Open and closed conformations of ecGlpG structures are compared with each other and with hiGlpG, surveying differences in hydrophobic interactions within loop 5 and helix 5. Furthermore, a comparison of the ecGlpG and hiGlpG structures reveals differences in loop 4. Overall, less variation is observed in loop 4, suggesting this region acts as an anchor for the substrate gate. Functional and regulatory implications of these variations are discussed.

Structural analysis of the Na+/H+ exchanger isoform 1 (NHE1) using the divide and conquer approachThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 189-199 ◽  
Author(s):  
Brian L. Lee ◽  
Brian D. Sykes ◽  
Larry Fliegel
Keyword(s):  
Crystal Structure ◽  
Membrane Protein ◽  
Peer Review Process ◽  
Divide And Conquer ◽  
Transmembrane Helices ◽  
Transmembrane Segments ◽  
Sodium Proton Exchanger ◽  
Attractive Therapeutic Target ◽  
Health And Disease ◽  
Selection Of

The sodium/proton exchanger isoform 1 (NHE1) is an ubiquitous plasma membrane protein that regulates intracellular pH by removing excess intracellular acid. NHE1 is important in heart disease and cancer, making it an attractive therapeutic target. Although much is known about the function of NHE1, current structural knowledge of NHE1 is limited to two conflicting topology models: a low-resolution molecular envelope from electron microscopy, and comparison with a crystal structure of a bacterial homologue, NhaA. Our laboratory has used high-resolution nuclear magnetic resonance (NMR) spectroscopy to investigate the structures of individual transmembrane helices of NHE1 — a divide and conquer approach to the study of this membrane protein. In this review, we discuss the structural and functional insights obtained from this approach in combination with functional data obtained from mutagenesis experiments on the protein. We also compare the known structure of NHE1 transmembrane segments with the structural and functional insights obtained from a bacterial sodium/proton exchanger homologue, NhaA. The structures of regions of the NHE1 protein that have been determined have both similarities and specific differences to the crystal structure of the NhaA protein. These have allowed insights into both the topology and the function of the NHE1 protein.

Expression and bioactivity of recombinant segments of human perforinThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

2007 ◽  
Vol 85 (2) ◽  
pp. 203-208 ◽  
Author(s):  
Hongmei Dong ◽  
Xiaohu Xu ◽  
Mohong Deng ◽  
Xiaojun Yu ◽  
Hu Zhao ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Target Genes ◽  
Review Process ◽  
Peer Review Process ◽  
Nitrilotriacetic Acid ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Expression Plasmids ◽  
Selection Of

The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28–349 aa), MAC2 (166–369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni–NTA, were ~80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28–349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.

scholarly journals Effect of Dimethyl Sulphoxide on the Expression of Nitrogen Fixation in Bacteria

1977 ◽  
Vol 30 (2) ◽  
pp. 141 ◽  
Author(s):  
Mary L Skotnicki ◽  
Barry G Rolfe
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
Energy Metabolism ◽  
Membrane Proteins ◽  
Klebsiella Pneumoniae ◽  
Dimethyl Sulphoxide ◽  
Rhizobium Trifolii ◽  
Nif Genes ◽  
E Coli ◽  
Selection Of

Storage in dimethyl sulphoxide (DMSO) of Escherichia coli K12 hybrids carrying nif+ genes from Klebsiella pneumoniae can result in selection of a defective nitrogen-fixing phenotype. Similar results are obtained with E. coli K12 hybrids containing the nitrogep-fixing capacity from Rhizobium trifolii. DMSO appears to affect particular inner membrane proteins associated with energy metabolism in E. coli K12 and four chromosomal regions (chID, chlG, his and unc) are associated with resistance to DMSO.

scholarly journals One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

2015 ◽  
Vol 113 (3) ◽  
pp. 608-613 ◽  
Author(s):  
Peter J. Yunker ◽  
Haruichi Asahara ◽  
Kuo-Chan Hung ◽  
Corey Landry ◽  
Laura R. Arriaga ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Self Assembly ◽  
Hydrophobic Interactions ◽  
Cell Communication ◽  
Transmembrane Helices ◽  
One Pot ◽  
Surface Receptors ◽  
Coupling Protein

Single-span membrane proteins (ssMPs) represent approximately one-half of all membrane proteins and play important roles in cellular communications. However, like all membrane proteins, ssMPs are prone to misfolding and aggregation because of the hydrophobicity of transmembrane helices, making them difficult to study using common aqueous solution-based approaches. Detergents and membrane mimetics can solubilize membrane proteins but do not always result in proper folding and functionality. Here, we use cell-free protein synthesis in the presence of oil drops to create a one-pot system for the synthesis, assembly, and display of functional ssMPs. Our studies suggest that oil drops prevent aggregation of some in vitro-synthesized ssMPs by allowing these ssMPs to localize on oil surfaces. We speculate that oil drops may provide a hydrophobic interior for cotranslational insertion of the transmembrane helices and a fluidic surface for proper assembly and display of the ectodomains. These functionalized oil drop surfaces could mimic cell surfaces and allow ssMPs to interact with cell surface receptors under an environment closest to cell–cell communication. Using this approach, we showed that apoptosis-inducing human transmembrane proteins, FasL and TRAIL, synthesized and displayed on oil drops induce apoptosis of cultured tumor cells. In addition, we take advantage of hydrophobic interactions of transmembrane helices to manipulate the assembly of ssMPs and create artificial clusters on oil drop surfaces. Thus, by coupling protein synthesis with self-assembly at the water–oil interface, we create a platform that can use recombinant ssMPs to communicate with cells.

Mis-trafficking of bicarbonate transporters: implications to human diseasesThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 157-177 ◽  
Author(s):  
Ensaf Y. Almomani ◽  
Carmen Y.S. Chu ◽  
Emmanuelle Cordat
Keyword(s):  
Peer Review Process ◽  
Waste Product ◽  
Gene Families ◽  
Acid Base Balance ◽  
Mammalian Cell Lines ◽  
Current State ◽  
Bicarbonate Transporters ◽  
Base Balance ◽  
Health And Disease ◽  
Selection Of

Bicarbonate is a waste product of mitochondrial respiration and one of the main buffers in the human body. Thus, bicarbonate transporters play an essential role in maintaining acid-base balance but also during fetal development as they ensure tight regulation of cytosolic and extracellular environments. Bicarbonate transporters belong to two gene families, SLC4A and SLC26A. Proteins from these two families are widely expressed, and thus mutations in their genes result in various diseases that affect bones, pancreas, reproduction, brain, kidneys, eyes, heart, thyroid, red blood cells, and lungs. In this minireview, we discuss the current state of knowledge regarding the effect of SLC4A and SLC26A mutants, with a special emphasis on mutants that have been studied in mammalian cell lines and how they correlate with phenotypes observed in mice models.

Topology models of anion exchanger 1 that incorporate the anti-parallel V-shaped motifs found in the EM structureThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 148-156 ◽  
Author(s):  
Teruhisa Hirai ◽  
Naotaka Hamasaki ◽  
Tomohiro Yamaguchi ◽  
Yohei Ikeda
Keyword(s):  
Anion Exchanger ◽  
Peer Review Process ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Dimensional Structure ◽  
Membrane Bilayer ◽  
Anion Exchanger 1 ◽  
Advantages And Disadvantages ◽  
Health And Disease ◽  
Selection Of

We recently published the three-dimensional structure of the membrane domain of human erythrocyte anion exchanger 1 (AE1) at 7.5 Å resolution, solved by electron crystallography. The structure exhibited distinctive anti-parallel V-shaped motifs, which protrude from the membrane bilayer on both sides. Similar motifs exist in the previously reported structure of a bacterial chloride channel (ClC)-type protein. Here, we propose two topology models of AE1 that reflect the anti-parallel V-shaped structural motifs. One is assumed to have structural similarity with the ClC protein and the other is only assumed to have internal repeats, as is often the case with transporters. Both models are consistent with most topological results reported thus far for AE1, each having advantages and disadvantages.

Anion exchanger 1 in red blood cells and kidney: Band 3’s in a podThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 106-114 ◽  
Author(s):  
Fiona Wu ◽  
Timothy J. Satchwell ◽  
Ashley M. Toye
Keyword(s):  
Review Process ◽  
Peer Review Process ◽  
Band 3 ◽  
Glomerular Filtration Barrier ◽  
Anion Exchanger 1 ◽  
Filtration Barrier ◽  
Kidney Glomerulus ◽  
Health And Disease ◽  
Distal Tubules ◽  
Selection Of

The bicarbonate/chloride exchanger 1 (AE1, Band 3) is abundantly expressed in the red blood cell membrane, where it is involved in gas exchange and functions as a major site of cytoskeletal attachment to the erythrocyte membrane. A truncated kidney isoform (kAE1) is highly expressed in type A intercalated cells of the distal tubules, where it is vital for urinary acidification. Recently, kAE1 has emerged as a novel physiologically significant protein in the kidney glomerulus. This minireview will discuss the known interactions of kAE1 in the podocytes and the possible mechanisms whereby this important multispanning membrane protein may contribute to the function of the glomerular filtration barrier and prevent proteinuria.

Investigating membrane protein dynamics in living cellsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

2006 ◽  
Vol 84 (6) ◽  
pp. 825-831 ◽  
Author(s):  
Ian R. Bates ◽  
Paul W. Wiseman ◽  
John W. Hanrahan
Keyword(s):  
Membrane Proteins ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Fluorescence Correlation ◽  
Transport Dynamics ◽  
Health And Disease ◽  
Image Correlation Spectroscopy ◽  
Selection Of

Live cell imaging is a powerful tool for understanding the function and regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based techniques for studying the transport dynamics of membrane proteins: fluorescence-correlation spectroscopy, image-correlation spectroscopy, fluorescence recovery after photobleaching, and single-particle and (or) molecule tracking. The advantages and limitations of each approach are illustrated using recent studies of an ion channel and cell adhesion molecules.

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