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Elizabeth E. Howell - One of the best experts on this subject based on the ideXlab platform.
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Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini.
Biochemistry, 2017Co-Authors: Purva P. Bhojane, Michael R. Duff, Khushboo Bafna, Pratul K. Agarwal, Christopher B. Stanley, Elizabeth E. HowellAbstract:R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 A. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 A. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and r...
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Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N‑Termini
2017Co-Authors: Purva P. Bhojane, Michael R. Duff, Khushboo Bafna, Pratul Agarwal, Christopher Stanley, Elizabeth E. HowellAbstract:R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the Protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the Protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the Protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated
Purva P. Bhojane - One of the best experts on this subject based on the ideXlab platform.
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Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini.
Biochemistry, 2017Co-Authors: Purva P. Bhojane, Michael R. Duff, Khushboo Bafna, Pratul K. Agarwal, Christopher B. Stanley, Elizabeth E. HowellAbstract:R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 A. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 A. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and r...
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Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N‑Termini
2017Co-Authors: Purva P. Bhojane, Michael R. Duff, Khushboo Bafna, Pratul Agarwal, Christopher Stanley, Elizabeth E. HowellAbstract:R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the Protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the Protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the Protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated
Almundher Almaawali - One of the best experts on this subject based on the ideXlab platform.
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homozygosity for farsb mutation leads to phe trna synthetase related disease of growth restriction brain calcification and interstitial lung disease
Human Mutation, 2018Co-Authors: Fahad Zadjali, Aida Alyahyaee, Maryam Alnabhani, Saif Almubaihsi, Arunodaya R Gujjar, Sameer Raniga, Almundher AlmaawaliAbstract:Aminoacyl-tRNA synthetases (ARSs) canonical function is to conjugate specific amino acids to cognate tRNA that are required for the first step of Protein synthesis. Genetic mutations that cause dysfunction or absence of ARSs result in various neurodevelopmental disorders. The human phenylalanine-tRNA synthetase (PheRS) is a Tetrameric Protein made of two subunits coded by FARSA gene and two subunits coded by FARSB gene. We describe eight affected individuals from an extended family with a multisystemic recessive disease manifest as a significant growth restriction, brain calcifications, and interstitial lung disease. Genome-wide linkage analysis and whole exome sequencing identified homozygosity for a FARSB mutation (NM_005687.4:c.853G > A:p.Glu285Lys) that co-segregate with the disease and likely cause loss-of-function. This study further implicates FARSB mutations in a multisystem, recessive, neurodevelopmental phenotype that share clinical features with the previously known aminoacyl-tRNA synthetase-related diseases.
Rui M. M. Brito - One of the best experts on this subject based on the ideXlab platform.
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The V30M Amyloidogenic Mutation Decreases the Rate of Refolding Kinetics of the Tetrameric Protein Transthyretin
Spectroscopy: An International Journal, 2012Co-Authors: Catarina S. H. Jesus, Daniela C. Vaz, Maria João Saraiva, Rui M. M. BritoAbstract:Transthyretin (TTR) is a homoTetrameric Protein implicated in several amyloid diseases. The mechanism by which TTR is converted into elongated fibrillar assemblies has been extensively investigated, and numerous studies showed that dissociation of the native Tetrameric structure into partially unfolded monomeric species precedes amyloid formation. The small differences observed in the crystal structures of different TTR variants, as well as the thermodynamics and kinetics of tetramer dissociation, do not seem to completely justify the amyloidogenic potential of different TTR variants. With this in mind, we have studied the refolding kinetics of WT-TTR and its most common amyloidogenic variant V30M-TTR, monitoring changes in intrinsic tryptophan fluorescence at different urea and Protein concentrations. Our results demonstrate that the in vitro refolding mechanisms of WT- and V30M-TTR are similar, involving a dimeric intermediate. However, there are large differences in the refolding rate constants for the two variants, specially close to physiological conditions. Interestingly, tetramer formation occurs at a much slower rate in the amyloidogenic variant V30M-TTR than in WT-TTR, which in the in vivo setting may promote the accumulation of monomeric species in the extracellular environment, resulting in higher susceptibility for aggregation and amyloid formation instead of spontaneous refolding.
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the Tetrameric Protein transthyretin dissociates to a non native monomer in solution a novel model for amyloidogenesis
Journal of Biological Chemistry, 1999Co-Authors: Alexandre Quintas, Maria João Saraiva, Rui M. M. BritoAbstract:Abstract In amyloidosis, normally innocuous soluble Proteins polymerize to form insoluble fibrils. Amyloid fibril formation and deposition have been associated with a wide range of diseases, including spongiform encephalopathies, Alzheimer's disease, and familial amyloid polyneuropathies (FAP). In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homoTetrameric plasma Protein implicated in the transport of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, and L55P-TTR is the variant associated with the most aggressive form of FAP. Recently, we reported that TTR dissociates to a monomeric species at pH 7.0 and nearly physiological ionic strengths (Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) FEBS Lett.418, 297–300). Here, we show that the tetramer dissociation is apparently irreversible; and based on intrinsic tryptophan fluorescence and fluorescence quenching experiments, we show that the monomeric species formed upon tetramer dissociation is non-native. We also show, based on 1-anilino-8-naph-thalenesulfonate binding studies, that this monomeric species appears not to behave like a molten globule. These data allowed us to propose a model for TTR amyloidogenesis based on tetramer dissociation occurring naturally under commonly observed physiological solution conditions.
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The Tetrameric Protein Transthyretin Dissociates to a Non-native Monomer in Solution
1999Co-Authors: Alexandre Quintas, M. Saraivai, Rui M. M. BritoAbstract:In amyloidosis, normally innocuous soluble Proteinspolymerize to form insoluble fibrils. Amyloid fibril for-mation and deposition have been associated with a widerange of diseases, including spongiform encephalopa-thies, Alzheimer’s disease, and familial amyloid poly-neuropathies (FAP). In certain forms of FAP, the amy-loid fibrils are mostly constituted by variants oftransthyretin (TTR), a homoTetrameric plasma Proteinimplicated in the transport of thyroxine and retinol. Themost common amyloidogenic TTR variant is V30M-TTR,and L55P-TTR is the variant associated with the mostaggressive form of FAP. Recently, we reported that TTRdissociates to a monomeric species at pH 7.0 and nearlyphysiological ionic strengths (Quintas, A., Saraiva, M. J.,and Brito, R. M. (1997)
Yuval Shoham - One of the best experts on this subject based on the ideXlab platform.
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Crystallization and preliminary X-ray analysis of family 39 β-d-xylosidase from Geobacillus stearothermophilus T-6
Acta Crystallographica Section D Biological Crystallography, 2004Co-Authors: Mirjam Czjzek, Tsafrir Bravman, Bernard Henrissat, Yuval ShohamAbstract:beta-D-Xylosidases (EC 3.2.1.37) are hemicellulases that hydrolyze short xylooligosaccharides into single xylose units. In this study, the crystallization and preliminary X-ray analysis of the beta-D-xylosidase (XynB1) from Geobacillus stearothermophilus T-6, a family 39 glycoside hydrolase, are described. XynB1 is a Tetrameric Protein consisting of four identical subunits of 503 amino acids and with a calculated molecular weight of 58 001 Da. Both the native and the selenomethionine-containing XynB1 were crystallized by the hanging-drop vapour-diffusion method and the crystals were found to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 92.7, b = 165.7, c = 311.0 A. The native crystals diffracted X-rays to a resolution of 2.1 A.
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Crystallization and preliminary X-ray analysis of family 39 beta-D-xylosidase from Geobacillus stearothermophilus T-6.
Acta crystallographica. Section D Biological crystallography, 2004Co-Authors: Mirjam Czjzek, Tsafrir Bravman, Bernard Henrissat, Yuval ShohamAbstract:beta-D-Xylosidases (EC 3.2.1.37) are hemicellulases that hydrolyze short xylooligosaccharides into single xylose units. In this study, the crystallization and preliminary X-ray analysis of the beta-D-xylosidase (XynB1) from Geobacillus stearothermophilus T-6, a family 39 glycoside hydrolase, are described. XynB1 is a Tetrameric Protein consisting of four identical subunits of 503 amino acids and with a calculated molecular weight of 58 001 Da. Both the native and the selenomethionine-containing XynB1 were crystallized by the hanging-drop vapour-diffusion method and the crystals were found to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 92.7, b = 165.7, c = 311.0 A. The native crystals diffracted X-rays to a resolution of 2.1 A.
