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A fragment-based approach for ligand binding affinity and selectivity for the liver X receptor beta

Salum, Lívia de Barros; Andricopulo, Adriano Defini; Honorio, Káthia Maria
Fonte: Elsevier Science INC; New York Publicador: Elsevier Science INC; New York
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
65.97%
Selective modulation of liver X receptor beta (LXR beta) has been recognized as an important approach to prevent or reverse the atherosclerotic process. In the present work, we have developed robust conformation-independent fragment-based quantitative structure-activity and structure-selectivity relationship models for a series of quinolines and cinnolines as potent modulators of the two LXR sub-types. The generated models were then used to predict the potency of an external test set and the predicted values were in good agreement with the experimental results, indicating the potential of the models for untested compounds. The final 2D molecular recognition patterns obtained were integrated to 3D structure-based molecular modeling studies to provide useful insights into the chemical and structural determinants for increased LXR beta binding affinity and selectivity. (C) 2011 Elsevier Inc. All rights reserved.; FAPESP (The State of Sao Paulo Research Foundation); FAPESP (The State of Sao Paulo Research Foundation); CNPq (The National Council for Scientific and Technological Development); CNPq (The National Council for Scientific and Technological Development); CAPES (Coordination for the Improvement of High Education Personnel), Brazil; CAPES (Coordination for the Improvement of High Education Personnel)...

Impedance-derived electrochemical capacitance spectroscopy for the evaluation of lectin-glycoprotein binding affinity

Santos, Adriano; Carvalho, Fernanda C.; Roque-Barreira, Maria-Cristina; Bueno, Paulo R.
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 102-105
Português
Relevância na Pesquisa
66.09%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Processo FAPESP: 12-22820-7; Characterization of lectin-carbohydrate binding using label-free methods such as impedance-derived electrochemical capacitance spectroscopy (ECS) is desirable to evaluate specific interactions, for example, ArtinM lectin and horseradish peroxidase (HRP) glycoprotein, used here as a model for proteincarbohydrate binding affinity. An electroactive molecular film comprising alkyl ferrocene as a redox probe and ArtinM as a carbohydrate receptive center to target HRP was successfully used to determine the binding affinity between ArtinM and HRP. The redox capacitance, a transducer signal associated with the alkyl ferrocene centers, was obtained by ECS and used in the Langmuir adsorption model to obtain the affinity constant (1.6 +/- 0.6) x 10(8) L mol(-1). The results shown herein suggest the feasibility of ECS application for lectin glycoarray characterization. (C) 2014 Elsevier B.V. All rights reserved.

2D and 3D QSAR studies of the receptor binding affinity of progestins

Veras,Lea da Silva; Arakawa,Masamoto; Funatsu,Kimito; Takahata,Yuji
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2010 Português
Relevância na Pesquisa
66%
A 2D QSAR analysis with three descriptors of binding affinity to human cytosol receptor was performed. The set of twenty-three progestins was divided into a training set of sixteen molecules and a test set of seven molecules. The quantum chemical RM1 semiempirical method was used to calculate geometry and some molecular properties. DRAGON software was also use to produce descriptors. MobyDigs software was used to select descriptors and build QSAR models. The best 2D QSAR model was constructed for the training set with multiple linear regression (MLR) using three descriptors , PW2, Mor15m, and GAP-10, resulting in r² = 0.866, q² = 0.805, q²boot = 0.723, q²ext = 0.666. A set of nine additional progestins that were not used for model building was used for external validation resulting q²ext = 0.403. The QSAR model was also validated by RQK fitness functions. It was shown to satisfy all the required criteria for validation. Two 3D QSAR models were built, first, to estimate predictive power, second, to analyze it. The predictive power of the 3D QSAR obtained with the nine external validation compounds was q²ext = 0.476. Based upon the graphical representation of PLS regression coefficients corresponding to steric and electrostatic interactions...

Age-associated mitochondrial oxidative decay: Improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-l- carnitine and/or R-α-lipoic acid

Liu, Jiankang; Killilea, David W.; Ames, Bruce N.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 19/02/2002 Português
Relevância na Pesquisa
46.18%
We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl-l-carnitine (ALCAR) and/or the mitochondrial antioxidant precursor R-α-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However, in vitro treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity...

Complex formation between CREB and Tax enhances the binding affinity of CREB for the human T-cell leukemia virus type 1 21-base-pair repeats.

Yin, M J; Gaynor, R B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1996 Português
Relevância na Pesquisa
46.18%
The regulation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is dependent on three cis-acting elements, known as the 21-bp repeats, in the long terminal repeat. Each of the 21-bp repeats contains a nonpalindromic cyclic AMP response element (CRE) sequence which is capable of binding members of the ATF/CREB family of transcription factors. The HTLV-1 transactivator protein Tax is able to markedly stimulate the in vitro binding of CREB to the CRE sites present in each of the 21-bp repeats but not to CRE sites present in cellular promoters. The ability to Tax to stimulate CREB binding to different CRE sites correlates with the ability of Tax to activate gene expression from these sites. We wished to determine how sequence differences between the somatostatin CRE and the 21-bp repeat were involved in this different response to Tax. Scatchard analysis indicated that CREB bound to the somatostatin CRE with a single class of high-affinity binding while CREB bound to the 21-bp repeats with a biphasic binding pattern, indicating the presence of both low- and high-affinity binding. Tax increased the affinity of CREB binding but not that of another ATF/CREB protein, CREB2, to the 21-bp repeat. However, Tax did not increase affinity of binding of CREB to the somatostatin CRE. To determine the mechanism by which Tax increased dCREB binding affinity...

Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes.

Koo, C; Lefkowitz, R J; Snyderman, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1983 Português
Relevância na Pesquisa
46.18%
The oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte (PMN) membranes exists in two affinity states. Since guanine nucleotides regulate the binding affinity and transductional activity of several other types of receptors, we examined the effect of nucleotides on the binding of N-formyl-methionyl peptides to their receptors on human PMN membranes. The addition of guanylylimidodiphosphate (0.1 mM), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMN membrane preparations reduced the fraction of high-affinity receptors detected in equilibrium binding studies from 21.3 +/- 0.13 to 11.8 +/- 0.05% (P less than 0.03), without altering the binding affinities. Since the total number of receptors remained unchanged, the effect of guanylylimidodiphosphate was to convert a portion of the receptors from the high-affinity state to the low-affinity state. At the maximal concentration of guanine nucleotide tested, approximately 50% of the high-affinity sites were converted to low-affinity sites. The findings obtained by equilibrium binding were supported by kinetic studies since the dissociation of the radiolabeled oligopeptide chemoattractant N-formyl-methionyl-leucyl-[3H]phenylalanine from PMN membranes was accelerated in the presence of guanine nucleotide. The effect of guanine nucleotides was reversed upon washing...

5-HT1A receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

Watson, J; Collin, L; Ho, M; Riley, G; Scott, C; Selkirk, J V; Price, G W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/2000 Português
Relevância na Pesquisa
46.18%
It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT1A receptors.Saturation studies using [3H]-8-OH-DPAT and [3H]-MPPF revealed a single, high affinity site (KD∼1 nM) in HEK293 cells expressing human 5-HT1A receptors and rat cortex. In recombinant cells, [3H]-MPPF labelled 3–4 fold more sites than [3H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [3H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT1A receptors but did not bind to native tissue 5-HT1A receptors. These data suggest that, in transfected HEK293 cells, human 5-HT1A receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state.Receptor agonists inhibited [3H]-MPPF binding from recombinant 5-HT1A receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [3H]-8-OH-DPAT and [3H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [3H]-MPPF and [3H]-8-OH-DPAT in a monophasic manner.Functional evaluation of compounds...

CD8 T cell autoreactivity to preproinsulin epitopes with very low human leucocyte antigen class I binding affinity

Abreu, J R F; Martina, S; Verrijn Stuart, A A; Fillié, Y E; Franken, K L M C; Drijfhout, J W; Roep, B O
Fonte: Blackwell Science Inc Publicador: Blackwell Science Inc
Tipo: Artigo de Revista Científica
Publicado em /10/2012 Português
Relevância na Pesquisa
46.18%
Beta cells presenting islet epitopes are recognized and destroyed by autoreactive CD8 T cells in type 1 diabetes. These islet-specific T cells are believed to react with epitopes binding with high affinity to human leucocyte antigen (HLA) expressed on beta cells. However, this assumption might be flawed in case of islet autoimmunity. We evaluated T cell recognition of the complete array of preproinsulin (PPI) peptides with regard to HLA binding affinity and T cell recognition. In a comprehensive approach, 203 overlapping 9–10mer PPI peptides were tested for HLA-A2 binding and subjected to binding algorithms. Subsequently, a high-throughput assay was employed to detect PPI-specific T cells in patient blood, in which conditional HLA ligands were destabilized by ultraviolet irradiation and HLA molecules refolded with arrays of PPI peptides, followed by quantum-dot labelling and T cell staining. Analysis of patient blood revealed high frequencies of CD8 T cells recognizing very low HLA binding peptides. Of 28 peptides binding to HLA-A2, a majority was predicted not to bind. Unpredicted peptides bound mainly with low affinities. HLA binding affinity and immunogenicity may not correlate in autoimmunity. Algorithms used to predict high-affinity HLA peptide binders discount the majority of low-affinity HLA binding epitopes. Appreciation that peptides binding HLA with very low affinity can act as targets of autoreactive T cells may help to understand loss of tolerance and disease pathogenesis and possibly point to tissue-specific immune intervention targets.

Prediction of Glycerol-Effect on Antigen-Antibody Binding Affinity from Molecular Dynamics Simulations

Vagenende, Vincent; Yap, Miranda G.S.; Trout, Bernhardt L.
Fonte: MIT - Massachusetts Institute of Technology Publicador: MIT - Massachusetts Institute of Technology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
66.04%
Many biological and biotechnological processes are controlled by protein-protein interactions in solution. In order to understand, predict and optimize such processes, it is valuable to understand how additives such as salts, sugars, polyols and denaturants affect protein-protein interactions. Currently, no methodology to foretell the effect of additives on protein-protein interactions has been established and frequently and extensive empirical screening to identify additives beneficial to the protein process is resorted to. In this work, we developed a methodology enabling the prediction of the additive-effect on the protein reaction equilibrium. The only prerequisite is that the atomic structure of the protein reactants and products are known. The methodology is based on the thermodynamic model for preferential interactions and makes use of molecular dynamics simulations to gauge additive-protein interactions. In order to validate our methodology, the change in binding affinity of the antibody fragment Y32S Fv D1.3 for lysozyme in the presence of varying glycerol concentrations is being calculated and the results will be compared with experimental data from literature. Finally, our methodology will be used to predict the glycerol effect on the binding affinity of wild type Fv D1.3 and various mutants.; Singapore-MIT Alliance (SMA)

The Mechanistic roles of reverse transcriptase dNTP binding affinity and the central poly purine tract in achieving effective transduction efficiency

Skasko, Mark (1981 - ); Kim, Baek
Fonte: Universidade de Rochester Publicador: Universidade de Rochester
Tipo: Tese de Doutorado Formato: Number of Pages:xv, 219 leaves
Português
Relevância na Pesquisa
66.24%
Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2008.; The previous enzymatic characterization of wild type Human Immunodeficiency Virus-1 (HIV-1) reverse transcriptase (RT) and mutant RTs, Q151N and V148I, clearly demonstrated the contribution of RT dNTP binding affinity (Kd) to the completion of DNA synthesis at low dNTP concentrations. The two mutant RTs, Q151N and V148I, were shown to have 234.2 and 25.6-fold lower binding affinity to the incoming dNTP, respectively. The follow-up experiments examined the cell-type specificity of HIV-1 vectors encoding these mutant RTs and demonstrated that while wild type vector could successfully transduce both high (i.e. CD4+ T cells) and low (i.e. macrophages) dNTP concentration cell-types, the mutant vectors’ ability to transduce low dNTP concentration cell-types was severely impaired. These data suggest that Kd of RT is one of the mechanistic elements involved in the target cell-type specificity of HIV-1. The presented collection of work describes two more systems that further characterize the role of Kd in retroviral celltype specificity as well as a potential novel role of the HIV-1 central polypurine tract (cPPT) in rescuing replication and transduction in viral vectors with enzymatically impaired RTs. First we compared the oncoretroviral RT of Murine Leukemia Virus (MuLV)...

A Competition Study on Copper-binding Affinity of SCO protein

Xu, Shuai
Fonte: Quens University Publicador: Quens University
Tipo: Project
Português
Relevância na Pesquisa
56.06%
Cytochrome c oxidase catalyzes the reduction of molecular oxygen to water and contributes to the electrochemical gradient by translocating protons across the membrane. The SCO protein (for Synthesis of Cytochrome c Oxidase) is proposed to be an important assembly factor in biogenesis of the oxidase. Particularly, SCO has been demonstrated to function as a metallochaperone that receives copper ions from an upstream copper source in the cell and subsequently delivers them to the CuA centre in subunit II of cytochrome c oxidase. However, the SCO protein binds copper ions tightly and forms a stable complex in vitro that is extremely difficult to dissociate. Direct titration and differential scanning calorimetry, for example, have demonstrated a tight binding between SCO and Cu(II). Nonetheless, the reported dissociation constant KD falls in a wide range from 65 nM to 3.5 pM. In this study, binding affinities of Bacillus subtilis SCO (BsSCO) for both Cu(II) and Cu(I) ions were quantitatively estimated via competition with various copper ion ligands and chelators. Ethylenediamine tetraacetic acid (EDTA) was used as a competitor to BsSCO. In this case, BsSCO is able to compete with the chelator for copper-binding, which binds Cu(II) with KD ~ 3.1 x 10-16 M (i.e....

A Competition Study on Copper-binding Affinity of SCO protein

Xu, Shuai
Fonte: Quens University Publicador: Quens University
Tipo: Project
Português
Relevância na Pesquisa
56.06%
Cytochrome c oxidase catalyzes the reduction of molecular oxygen to water and contributes to the electrochemical gradient by translocating protons across the membrane. The SCO protein (for Synthesis of Cytochrome c Oxidase) is proposed to be an important assembly factor in biogenesis of the oxidase. Particularly, SCO has been demonstrated to function as a metallochaperone that receives copper ions from an upstream copper source in the cell and subsequently delivers them to the CuA centre in subunit II of cytochrome c oxidase. However, the SCO protein binds copper ions tightly and forms a stable complex in vitro that is extremely difficult to dissociate. Direct titration and differential scanning calorimetry, for example, have demonstrated a tight binding between SCO and Cu(II). Nonetheless, the reported dissociation constant KD falls in a wide range from 65 nM to 3.5 pM. In this study, binding affinities of Bacillus subtilis SCO (BsSCO) for both Cu(II) and Cu(I) ions were quantitatively estimated via competition with various copper ion ligands and chelators. Ethylenediamine tetraacetic acid (EDTA) was used as a competitor to BsSCO. In this case, BsSCO is able to compete with the chelator for copper-binding, which binds Cu(II) with KD ~ 3.1 x 10-16 M (i.e....

A novel androgen receptor mutant, A748T, exhibits hormone concentration-dependent defects in nuclear accumulation and activity despite normal hormone-binding affinity

James, A.; Agoulnik, I.; Harris, J.; Buchanan, G.; Tilley, W.; Marcelli, M.; Lamb, D.; Weigel, N.
Fonte: Endocrine Soc Publicador: Endocrine Soc
Tipo: Artigo de Revista Científica
Publicado em //2002 Português
Relevância na Pesquisa
55.95%
Functional analysis of androgen receptor (AR) gene mutations isolated from prostate cancer has led to the identification of residues that play important roles in the structure and function of the receptor. Here we report the characteristics of a novel AR mutation A748T located in helix 5 of the ligand-binding domain, which was identified in metastatic prostate cancer. Despite a normal hormone-binding affinity, A748T causes hormone concentration-dependent defects in nuclear accumulation and transcriptional activation. Moreover, when equivalent amounts of DNA are transfected, the mutant is expressed at much lower levels than the wild-type AR (ARWT). Treatment with geldanamycin to disrupt receptor-heat shock protein complexes rapidly decreases the levels of ARWT but not A748T, suggesting that the lower expression and rapid degradation rate of A748T is due to weaker interactions with heat shock proteins. Further analysis revealed that hormone dissociates from A748T five times faster than from ARWT. Loss of the ability to form stable amino/carboxyl-terminal interactions causes accelerated dissociation rates in some AR mutants. However, A748T exhibits normal amino/carboxyl-terminal interactions at high hormone concentrations, suggesting that the mutation alters interactions with ligand. Consistent with this conclusion...

Bindung und Wirkung von Modulatoren der ATP-empfindlichen Kaliumkanäle; Binding and effect of KATP channel modulators

Stephan, Damian Makary
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
Relevância na Pesquisa
46.22%
1ATP-sensitive K+-Kanäle (KATP-Kanäle) sind Heterooktamere aus vier porenbildenden Untereinheiten Kir (2 Subtypen: Kir6.1 und Kir6.2) und vier regulatorischen Untereinheiten SUR (2 Subtypen: SUR1 und SUR2 mit zwei Hauptspleißvarianten 2A und 2B). Sie kommen, unterschiedlich zusammensetzt, in vielen Geweben (z. B. Kir6.2/SUR1 in beta-Zellen des Pankreas, Kir6.2/SUR2A im Myokard, Kir6.1/SUR2B im glatten Gefäßmuskel) vor und regulieren dort wichtige physiologische Funktionen. Sie sind Zielstrukturen für pharmakologische Modulatoren wie die Kanal-Blocker (Sulfonylharnstoffe und Glinide) und Öffner (z.B. Bimakalim). In dieser Arbeit wurden folgende Schwerpunkte bearbeitet: Die Selektivität von Sulfonylharnstoffen (SU) und Gliniden in Bindung und Wirkung für die drei wichtigsten KATP-Kanalsubtypen Kir6.2/SUR1 (Pankreas), Kir6.2/SUR2A (Myokard) und Kir6.1/SUR2B (Gefäß) Der Einfluss der Koexpression mit Kir6.x auf die Bindung von SU und Gliniden an SUR1 Der Einfluss der Mutation Y1206S und der Koexpression mit Kir6.x auf den Sulfonylharnstoffrezeptor SUR2A Der Zugang der KATP-Kanal-Öffner vom Benzopyrantyp an ihre Bindungsstelle Die Affinität ausgewählter SU und Glinide zu den rekombinanten Kir6.2/SUR1- und Kir6.2/SUR2A-Kanälen wurde mittels Verdrängung des 3H-Glibenclamids (3H-GBC) an intakten HEK-Zellen bei 37ºC bestimmt. Dabei wurden keine nennenswerten Unterschiede in der Selektivität zwischen den reinen A-Liganden (kurzkettiger SU Glibornurid und das D-Phenylalanin-Derivat Nateglinid) und den A+B-Liganden (langkettige SU: GBC...

Examining the Use of Homology Models in Predicting Kinase Binding Affinity

Chyan, Jeffrey
Fonte: Universidade Rice Publicador: Universidade Rice
Português
Relevância na Pesquisa
66.11%
Drug design is a difficult and multi-faceted problem that has led to extensive interdiscplinary work in the field of computational biology. In recent years, several computational methods have emerged. The overall goal of computational algorithms is to narrow down the number of leads that will be further considered for laboratory experimentation and clinical studies. Much of current drug design focuses on a family of proteins called kinases because they play a pivotal role in many of the cell signaling pathways in the human body. Drugs need to be designed such that they bind to specific kinases in the human kinome inhibiting kinase functions that can be causing various diseases such as cancer. It is important for drugs to have high specificity inhibiting only certain kinases avoiding undesirable effects on the human body. Computational prediction methods can accomplish this complex task by doing a comparative analysis on the binding site of kinases both in sequence and structure to predict binding affinity with potential drugs. However, computational methods depend on existing protein data to make predictions. There is a lack of structural protein data relative to known proteins and protein sequences. A potential solution to the the lack of information is to use computationally generated structural data called homology models. This thesis introduces a framework for the integration of homology models with CCORPS...

Removal of the four c-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley b-amylase

Ma, Y.; Eglinton, J.; Evans, D.; Logue, S.; Langridge, P.
Fonte: Amer Chemical Soc Publicador: Amer Chemical Soc
Tipo: Artigo de Revista Científica
Publicado em //2000 Português
Relevância na Pesquisa
56.09%
Barley beta-amylase undergoes proteolytic cleavage in the C-terminal region after germination. The implication of the cleavage in the enzyme's characteristics is unclear. With purified native beta-amylases from both mature barley grain and germinated barley, we found that the beta-amylase from germinated barley had significantly higher thermostability and substrate binding affinity for starch than that from mature barley grain. To better understand the effect of the proteolytic cleavage on the enzyme's thermostability and substrate binding affinity for starch, recombinant barley beta-amylases with specific deletions at the C-terminal tail were generated. The complete deletion of the four C-terminal glycine-rich repeats significantly increased the enzyme's thermostability, but an incomplete deletion with one repeat remaining did not change the thermostability. Although different C-terminal deletions affect the thermostability differently, they all increased the enzyme's affinity for starch. The possible reasons for the increased thermostability and substrate binding affinity, due to the removal of the four C-terminal glycine-rich repeats, are discussed in terms of the three-dimensional structure of beta-amylase.; Yue F. Ma, Jason K. Eglinton...

Mutations of barley b-amylase that improve substrate-binding affinity and thermostability

Ma, Y.; Evans, D.; Logue, S.; Langridge, P.
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Publicado em //2001 Português
Relevância na Pesquisa
66%
Three allelic forms of barley β-amylase (Sd1, Sd2H and Sd2L) exhibit different thermostability and kinetic properties. These differences critically influence the malting quality of barley varieties. To understand the molecular basis for the different properties of these three allelic forms, Sd1 and Sd2L β-amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis. The results showed that an R115C mutation is responsible for the difference in kinetic properties. This substitution resulted in an additional hydrogen bond which may create a more favourable environment for substrate-binding. The different thermostabilities of the β-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability index T₅₀ by 1.9°C and 2.1°C, respectively. The increased thermostability associated with these two mutations may be due to relief of steric strain and the interaction of the protein surface with solvent water. Although both V233A and L347S mutations increased thermostability, they affected the thermostability in different ways. The replacement of L347 by serine seems to increase the thermostability by slowing thermal unfolding of the protein during heating...

A Competition Study on Copper-binding Affinity of SCO protein

Xu, SHUAI
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado
Português
Relevância na Pesquisa
56.06%
Cytochrome c oxidase catalyzes the reduction of molecular oxygen to water and contributes to the electrochemical gradient by translocating protons across the membrane. The SCO protein (for Synthesis of Cytochrome c Oxidase) is proposed to be an important assembly factor in biogenesis of the oxidase. Particularly, SCO has been demonstrated to function as a metallochaperone that receives copper ions from an upstream copper source in the cell and subsequently delivers them to the CuA centre in subunit II of cytochrome c oxidase. However, the SCO protein binds copper ions tightly and forms a stable complex in vitro that is extremely difficult to dissociate. Direct titration and differential scanning calorimetry, for example, have demonstrated a tight binding between SCO and Cu(II). Nonetheless, the reported dissociation constant KD falls in a wide range from 65 nM to 3.5 pM. In this study, binding affinities of Bacillus subtilis SCO (BsSCO) for both Cu(II) and Cu(I) ions were quantitatively estimated via competition with various copper ion ligands and chelators. Ethylenediamine tetraacetic acid (EDTA) was used as a competitor to BsSCO. In this case, BsSCO is able to compete with the chelator for copper-binding, which binds Cu(II) with KD ~ 3.1 x 10-16 M (i.e....

The affinities for the two substrate water binding sites in the O2 evolving complex of Photosystem II vary independently during S-state turnover.

Hillier, Warwick; Wydrzynski, Thomas
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
56.06%
The first determinations of substrate water binding to the O2 evolving complex in photosystem II as a complete function of the S states have been made. H218O was rapidly injected into spinach thylakoid samples preset in either the S0, S1, S2, or S3 states, and the rate of 18O incorporation into the O2 produced was determined by time-resolved mass spectrometry. For measurements at m/e = 34 (i.e., for the 16O18O product), the rate of 18O incorporation in all S states shows biphasic kinetics, reflecting the binding of the two substrate water molecules to the catalytic site. The slow phase kinetics yield rate constants at 10 °C of 8 ± 2, 0.021 ± 0.002, 2.2 ± 0.3, and 1.9 ± 0.2 s-1 for the S0, S1, S2, and S3 states, respectively, while the fast phase kinetics yield a rate constant of 36.8 ± 1.9 s-1 for the S3 state but remain unresolvable (> 100 s-1) for the S0, S1, and S2 states. Comparisons of the 18O exchange rates reveal that the binding affinity for one of the substrate water molecules first increases during the S0 to S1 transition, then decreases during the S1 to S2 transition, but stays the same during the S2 to S3 transition, while the binding affinity for the second substrate water molecule undergoes at least a 5-fold increase on the S2 to S3 transition. These findings are discussed in terms of two independent Mn(III) substrate binding sites within the O2 evolving complex which are separate from the component that accumulates the oxidizing equivalents. One of the Mn(III) sites may only first bind a substrate water molecule during the S2 to S3 transition.

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis

Duggin, Iain; Matthews, Jacqueline; Dixon, Nicholas; Wake, R. Gerry; Mackay, Joel Peter
Fonte: American Society for Biochemistry and Molecular Biology Inc Publicador: American Society for Biochemistry and Molecular Biology Inc
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
46.2%
Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus...