Wild Type, Mutant Protein Unfolding and Phase Transition Detected by Single-Nanopore Recording

2012 ◽  
Vol 7 (4) ◽  
pp. 652-658 ◽  
Author(s):  
Céline Merstorf ◽  
Benjamin Cressiot ◽  
Manuela Pastoriza-Gallego ◽  
Abdelghani Oukhaled ◽  
Jean-Michel Betton ◽  
...  
Keyword(s):  
Phase Transition ◽  
Protein Unfolding ◽  
Mutant Protein ◽  
Wild Type ◽  
Type Mutant

scholarly journals Engineering Desiccation Tolerance inEscherichia coli

2000 ◽  
Vol 66 (4) ◽  
pp. 1680-1684 ◽  
Author(s):  
Daniela Billi ◽  
Deborah J. Wright ◽  
Richard F. Helm ◽  
Todd Prickett ◽  
Malcolm Potts ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phase Transition ◽  
Freeze Drying ◽  
Fold Increase ◽  
Inescherichia Coli ◽  
Vibration Frequencies ◽  
Wild Type ◽  
Air Drying ◽  
Phase Transition Temperatures ◽  
Cell Dehydration

ABSTRACT Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized inEscherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (PO stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.

Mechanisms for auto-inhibition and forced product release in glycine N-methyltransferase: crystal structures of wild-type, mutant R175K and S-adenosylhomocysteine-bound R175K enzymes

2000 ◽  
Vol 298 (1) ◽  
pp. 149-162 ◽  
Author(s):  
Yafei Huang ◽  
Junichi Komoto ◽  
Kiyoshi Konishi ◽  
Yoshimi Takata ◽  
Hirofumi Ogawa ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Wild Type ◽  
Product Release ◽  
Type Mutant

scholarly journals The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope.

1992 ◽  
Vol 119 (6) ◽  
pp. 1441-1449 ◽  
Author(s):  
R W Wozniak ◽  
G Blobel
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Immunofluorescence Microscopy ◽  
Nuclear Pore ◽  
Transmembrane Helices ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Membrane Domain ◽  
3T3 Cells ◽  
Type Mutant

The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.

scholarly journals Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-Crystallin on Chaperone Function and Anti-Apoptotic Activity

2021 ◽  
Vol 22 (19) ◽  
pp. 10771
Author(s):  
Sundararajan Mahalingam ◽  
Srabani Karmakar ◽  
Puttur Santhoshkumar ◽  
Krishna K. Sharma
Keyword(s):  
Deletion Mutant ◽  
Mutant Protein ◽  
Wild Type ◽  
Chaperone Function ◽  
Type Protein ◽  
Wild Type Protein ◽  
Cytotoxicity Studies ◽  
Αb Crystallin ◽  
Apoptotic Activity ◽  
Subunit Organization

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

scholarly journals Molecular Pathogenesis of Type I Congenital Plasminogen Deficiency: Expression of Recombinant Human Mutant Plasminogens in Mammalian Cells

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 183-190 ◽  
Author(s):  
Hiroyuki Azuma ◽  
Nobuaki Mima ◽  
Mitsuo Shirakawa ◽  
Kazumasa Miyamoto ◽  
Hiroshi Yamaguchi ◽  
...  
Keyword(s):  
Sodium Dodecyl ◽  
Resistant Mutant ◽  
Molecular Pathogenesis ◽  
Mutant Protein ◽  
Immunocytochemical Staining ◽  
Type I ◽  
Wild Type ◽  
Transport Pathway ◽  
Stable Transfectants ◽  
Plasminogen Deficiency

We previously reported the genetic abnormality in a Japanese family with type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation. To characterize the molecular pathogenesis of the disease in this family, we expressed recombinant human wild-type and mutant (rS572P) plasminogens in COS-1 cells. Activation-resistant wild-type and mutant plasminogen stable transfectants in CHO-K1 cells also were established. Transient transfection and metabolic labeling experiments followed by immunoprecipitation analysis showed that the mutant plasminogen was secreted from COS-1 cells in reduced amounts, compared with the wild type. Endo H digestion of the wild-type and mutant plasminogen showed no shift in their migrations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, indicating that both contain complex type oligosaccharide structures and could therefore be secreted. Furthermore, the secretion of activation-resistant mutant plasminogen was significantly reduced. Pulse-chase experiments and Northern blot analysis showed that the impaired secretion of the mutant plasminogen was the consequence of the accumulation of the mutant protein inside the cells but not of reduced plasminogen mRNA. Immunocytochemical staining of stable transfectants also revealed that CHO-K1 cells expressing the activation-resistant mutant plasminogen stained mainly in the perinuclear area, suggesting delayed processing of the mutant protein in the intracellular transport pathway. We conclude that the impaired secretion of mutant plasminogen, due to intracellular accumulation, is the molecular pathogenesis of type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation.

scholarly journals Single-tube genotyping for small insertion/deletion mutations: simultaneous identification of wild type, mutant and heterozygous alleles

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Bin Lin ◽  
Jing Sun ◽  
Iain D C Fraser
Keyword(s):  
Wild Type ◽  
Single Polymerase Chain Reaction ◽  
Deletion Mutations ◽  
Systematic Testing ◽  
Small Indels ◽  
Naturally Occurring ◽  
Induced Mutagenesis ◽  
Type Mutant ◽  
Polymerase Chain ◽  
Simultaneous Identification

Abstract Current methods of genotyping small insertion/deletion (indel) mutations are costly, laborious, and can be unreliable. To address this, we have developed a method for small indel genotyping in a single polymerase chain reaction, with wild-type, heterozygous and mutant alleles distinguishable by band pattern in routine agarose gel electrophoresis. We demonstrate this method with multiple genes to distinguish 10 bp, 4 bp and even 1 bp deletions from the wild type. Through systematic testing of numerous primer designs, we also propose guidelines for genotyping small indel mutations. Our method provides a convenient approach to genotyping small indels derived from clustered regularly interspaced short palindromic repeats-mediated gene editing, N-ethyl-N-nitrosourea induced mutagenesis or diagnosis of naturally occurring polymorphisms/mutations.

scholarly journals Molecular Pathogenesis of Type I Congenital Plasminogen Deficiency: Expression of Recombinant Human Mutant Plasminogens in Mammalian Cells

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 183-190 ◽  
Author(s):  
Hiroyuki Azuma ◽  
Nobuaki Mima ◽  
Mitsuo Shirakawa ◽  
Kazumasa Miyamoto ◽  
Hiroshi Yamaguchi ◽  
...  
Keyword(s):  
Sodium Dodecyl ◽  
Resistant Mutant ◽  
Molecular Pathogenesis ◽  
Mutant Protein ◽  
Immunocytochemical Staining ◽  
Type I ◽  
Wild Type ◽  
Transport Pathway ◽  
Stable Transfectants ◽  
Plasminogen Deficiency

Abstract We previously reported the genetic abnormality in a Japanese family with type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation. To characterize the molecular pathogenesis of the disease in this family, we expressed recombinant human wild-type and mutant (rS572P) plasminogens in COS-1 cells. Activation-resistant wild-type and mutant plasminogen stable transfectants in CHO-K1 cells also were established. Transient transfection and metabolic labeling experiments followed by immunoprecipitation analysis showed that the mutant plasminogen was secreted from COS-1 cells in reduced amounts, compared with the wild type. Endo H digestion of the wild-type and mutant plasminogen showed no shift in their migrations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, indicating that both contain complex type oligosaccharide structures and could therefore be secreted. Furthermore, the secretion of activation-resistant mutant plasminogen was significantly reduced. Pulse-chase experiments and Northern blot analysis showed that the impaired secretion of the mutant plasminogen was the consequence of the accumulation of the mutant protein inside the cells but not of reduced plasminogen mRNA. Immunocytochemical staining of stable transfectants also revealed that CHO-K1 cells expressing the activation-resistant mutant plasminogen stained mainly in the perinuclear area, suggesting delayed processing of the mutant protein in the intracellular transport pathway. We conclude that the impaired secretion of mutant plasminogen, due to intracellular accumulation, is the molecular pathogenesis of type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation.

How is KRAS testing associated with treatment and supportive care for patients with metastatic colorectal cancer? VA national assist project.

2013 ◽  
Vol 31 (31_suppl) ◽  
pp. 64-64
Author(s):  
Aparna Raj Parikh ◽  
Benjamin Kim ◽  
Philip Pantoja ◽  
Diana M. Tisnado ◽  
Sangeeta C Ahluwalia ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Supportive Care ◽  
Metastatic Colorectal Cancer ◽  
Systemic Therapy ◽  
Wild Type ◽  
Care Processes ◽  
Systemic Treatments ◽  
Intensity Of Care ◽  
Type Mutant ◽  
Eol Care

64 Background: EGFR monoclonal antibody (mAb) therapies improve quality of life and outcomes for metastatic colorectal cancer (mCRC), but only wild-type KRAS benefit from treatment. We evaluated KRAS testing and pharmacogenetic-guided treatment and supportive and end of life (EOL) care. Methods: Among a national random sample of 265 veterans diagnosed with mCRC in 2008 we evaluated KRAS testing, EGFR mAb therapy, supportive care using the Cancer Quality ASSIST indicators, and healthcare use. Three oncology nurses abstracted charts for care received 2008-2011. We linked chart to VA and Medicare administrative data and compared care received by KRAS testing and results. Results: 227/265 (85%) veterans died within 3 years and received an average of 48% of recommended supportive care processes. 96 / 265 (36%) underwent KRAS testing, of whom 41, 42, and 13 had wild-type, mutant, and indeterminant/unknown KRAS. 27/41 (66%) wild-type KRAS patients received an EGFR mAb; 18/45 (40%) patients receiving an EGFR mAb had mutant, indeterminant/unknown, or untested KRAS. KRAS testing was associated with increased systemic therapy but not differences in supportive care or intensity of care at the EOL. KRAS tested vs. not tested received hospice or palliative care (67 vs. 73%, p=0.55); any systemic therapy (96 vs. 40%, p<0.001) including new regimen in last month (0 vs. 6%); any chemotherapy in last 14 days of life (9 vs. 7%); and any acute care in last month of life (28 vs. 32%, p=0.78). Conclusions: KRAS testing was not performed for most veterans with mCRC in 2008, and EGFR mAb therapy was administered to many without wild-type KRAS. KRAS-tested patients were more likely to receive systemic treatments, and testing was not associated with greater intensity or inappropriate EOL care, and rates of such care were low among veterans.

scholarly journals Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4

2004 ◽  
Vol 164 (5) ◽  
pp. 701-715 ◽  
Author(s):  
Matthias Versele ◽  
Jeremy Thorner
Keyword(s):  
Wild Type ◽  
Gtp Hydrolysis ◽  
Filament Assembly ◽  
Gtp Binding ◽  
Type Mutant ◽  
Binding Residues ◽  
Septin Filament ◽  
Assembly Defect

Assembly at the mother–bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.

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