The Experts below are selected from a list of 59796 Experts worldwide ranked by ideXlab platform
Wlodzimierz Bujalowski - One of the best experts on this subject based on the ideXlab platform.
-
Interactions of the Escherichia coli DnaB helicase hexamer with the replication factor the DnaC protein. Effect of nucleotide cofactors and the ssDNA on protein-protein interactions and the topology of the complex.
Journal of Molecular Biology, 2003Co-Authors: Roberto Galletto, Maria J. Jezewska, Wlodzimierz BujalowskiAbstract:Abstract Quantitative studies of interactions between the Escherichia coli replication factor DnaC protein and the DnaB helicase have been performed using sedimentation velocity and Fluorescence Energy Transfer techniques. The applied novel analysis of the sedimentation data allows us to construct thermodynamic rigorous binding isotherms without any assumption as to the relationship between the observed molecular property of the complexes formed, the average sedimentation coefficient, or the degree of binding. Experiments have been performed with the fluorescein-modified DnaB helicase, which allows an exclusive monitoring of the DnaB–DnaC complex formation. The DnaC binding to the unmodified helicase has been characterized in competition experiments. The data establish that, in the presence of the ATP analog AMP-PNP, or ADP, a maximum of six DnaC monomers bind cooperatively to the DnaB hexamer. The positive cooperative interactions are limited to the two neighboring DnaC molecules. Analyses using a statistical thermodynamic hexagon model indicate that, under the solution conditions examined, the affinity is characterized by the intrinsic binding constant K=1.4(±0.5)×105 M−1 and cooperativity parameter σ=21±5. These data suggest strongly that the DnaC–DnaB complex exists in vivo as a mixture of complexes with a different number of bound DnaC molecules, although the complex with six DnaC molecules bound dominates the distribution. The DnaC nucleotide-binding site is not involved in the stabilization of the complex. Moreover, the hydrolysis of NTP bound to the helicase or the DnaC is not required for the release of the DnaC protein from the complex. The single-stranded DNA (ssDNA) bound to the helicase does not affect the DnaC protein binding. However, in the presence of the DNA, there is a significant difference in the energetics and structure of the ternary complex, DnaC–DnaB–ssDNA, formed in the presence of AMP-PNP as compared to ADP. The topology of the ternary complex DnaC–DnaB–ssDNA has been determined using the Fluorescence Energy Transfer method. In solution, the DnaC protein-binding site is located on the large 33 kDa domain of the DnaB helicase. The significance of the results in the functioning of the DnaB helicase–DnaC protein complex is discussed.
-
does single stranded dna pass through the inner channel of the protein hexamer in the complex with the escherichia coli dnab helicase Fluorescence Energy Transfer studies
Journal of Biological Chemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Danuta Bujalowska, Wlodzimierz BujalowskiAbstract:Abstract The structure of the complex of theEscherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the Fluorescence Energy Transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another Fluorescence label placed in different locations on the DNA substrates. Using the multiple Fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5′ arm of the replication fork, without ATP hydrolysis, the distance between the 5′ end of the arm and the 12-kDa domains of the hexamer (R = 47 A) is the same as in the complex with the isolated ssDNA oligomer (R = 47 A) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5′ arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2–3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free Energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.
-
functional and structural heterogeneity of the dna binding site of the escherichia coli primary replicative helicase dnab protein
Journal of Biological Chemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Wlodzimierz BujalowskiAbstract:Abstract The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative Fluorescence titration, centrifugation, and Fluorescence Energy Transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude ∼10 nucleotides. ssDNA affinity for the strong subsite is ∼3 orders of magnitude higher than that for the weak subsite. Fluorescence Energy Transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5′-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.
-
complex of escherichia coli primary replicative helicase dnab protein with a replication fork recognition and structure
Biochemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Wlodzimierz BujalowskiAbstract:Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous Fluorescence titration, Fluorescence Energy Transfer, ...
-
close proximity of tryptophan residues and atp binding site in escherichia coli primary replicative helicase dnab protein molecular topography of the enzyme
Journal of Biological Chemistry, 1994Co-Authors: Wlodzimierz Bujalowski, Malgorzata Maria KlonowskaAbstract:Abstract The binding of fluorescent nucleotide analogs to the Escherichia coli primary replicative helicase DnaB protein causes strong quenching of protein tryptophan Fluorescence. It results from the efficient Fluorescence Energy Transfer (E) from tryptophans to analogs bound in the nucleotide-binding site, indicating that protein tryptophans are "clustered" in close proximity to the binding site. This is in contrast to the lack of detectable Energy Transfer to the fluorescent single-stranded DNA (ssDNA) derivative, suggesting a distant separation between two function-linked structural elements of the enzyme, the nucleotide- and ssDNA-binding sites. The dependence of E upon the average number of bound nucleotides/DnaB hexamer is nonlinear, implying a larger separation between tryptophans and the bound nucleotide in the low affinity sites. Spectroscopic studies reveal that tryptophan residues are located on the surface of the DnaB helicase in a hydrophobic cleft, whereas the environment of the tyrosines is heterogeneous, with 6 out of 10 tyrosine residues located on the surface of the helicase. The efficiency of the Fluorescence Energy Transfer from the tyrosines to tryptophans suggests that the "centers of mass" of the residues are separated, possibly reflecting the separation of the nucleotide- and ssDNA-binding sites, with tyrosines constituting part of the ssDNA-binding region.
Maria J. Jezewska - One of the best experts on this subject based on the ideXlab platform.
-
Interactions of the Escherichia coli DnaB helicase hexamer with the replication factor the DnaC protein. Effect of nucleotide cofactors and the ssDNA on protein-protein interactions and the topology of the complex.
Journal of Molecular Biology, 2003Co-Authors: Roberto Galletto, Maria J. Jezewska, Wlodzimierz BujalowskiAbstract:Abstract Quantitative studies of interactions between the Escherichia coli replication factor DnaC protein and the DnaB helicase have been performed using sedimentation velocity and Fluorescence Energy Transfer techniques. The applied novel analysis of the sedimentation data allows us to construct thermodynamic rigorous binding isotherms without any assumption as to the relationship between the observed molecular property of the complexes formed, the average sedimentation coefficient, or the degree of binding. Experiments have been performed with the fluorescein-modified DnaB helicase, which allows an exclusive monitoring of the DnaB–DnaC complex formation. The DnaC binding to the unmodified helicase has been characterized in competition experiments. The data establish that, in the presence of the ATP analog AMP-PNP, or ADP, a maximum of six DnaC monomers bind cooperatively to the DnaB hexamer. The positive cooperative interactions are limited to the two neighboring DnaC molecules. Analyses using a statistical thermodynamic hexagon model indicate that, under the solution conditions examined, the affinity is characterized by the intrinsic binding constant K=1.4(±0.5)×105 M−1 and cooperativity parameter σ=21±5. These data suggest strongly that the DnaC–DnaB complex exists in vivo as a mixture of complexes with a different number of bound DnaC molecules, although the complex with six DnaC molecules bound dominates the distribution. The DnaC nucleotide-binding site is not involved in the stabilization of the complex. Moreover, the hydrolysis of NTP bound to the helicase or the DnaC is not required for the release of the DnaC protein from the complex. The single-stranded DNA (ssDNA) bound to the helicase does not affect the DnaC protein binding. However, in the presence of the DNA, there is a significant difference in the energetics and structure of the ternary complex, DnaC–DnaB–ssDNA, formed in the presence of AMP-PNP as compared to ADP. The topology of the ternary complex DnaC–DnaB–ssDNA has been determined using the Fluorescence Energy Transfer method. In solution, the DnaC protein-binding site is located on the large 33 kDa domain of the DnaB helicase. The significance of the results in the functioning of the DnaB helicase–DnaC protein complex is discussed.
-
does single stranded dna pass through the inner channel of the protein hexamer in the complex with the escherichia coli dnab helicase Fluorescence Energy Transfer studies
Journal of Biological Chemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Danuta Bujalowska, Wlodzimierz BujalowskiAbstract:Abstract The structure of the complex of theEscherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the Fluorescence Energy Transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another Fluorescence label placed in different locations on the DNA substrates. Using the multiple Fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5′ arm of the replication fork, without ATP hydrolysis, the distance between the 5′ end of the arm and the 12-kDa domains of the hexamer (R = 47 A) is the same as in the complex with the isolated ssDNA oligomer (R = 47 A) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5′ arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2–3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free Energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.
-
functional and structural heterogeneity of the dna binding site of the escherichia coli primary replicative helicase dnab protein
Journal of Biological Chemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Wlodzimierz BujalowskiAbstract:Abstract The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative Fluorescence titration, centrifugation, and Fluorescence Energy Transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude ∼10 nucleotides. ssDNA affinity for the strong subsite is ∼3 orders of magnitude higher than that for the weak subsite. Fluorescence Energy Transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5′-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.
-
complex of escherichia coli primary replicative helicase dnab protein with a replication fork recognition and structure
Biochemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Wlodzimierz BujalowskiAbstract:Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous Fluorescence titration, Fluorescence Energy Transfer, ...
Surendran Rajendran - One of the best experts on this subject based on the ideXlab platform.
-
does single stranded dna pass through the inner channel of the protein hexamer in the complex with the escherichia coli dnab helicase Fluorescence Energy Transfer studies
Journal of Biological Chemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Danuta Bujalowska, Wlodzimierz BujalowskiAbstract:Abstract The structure of the complex of theEscherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the Fluorescence Energy Transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another Fluorescence label placed in different locations on the DNA substrates. Using the multiple Fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5′ arm of the replication fork, without ATP hydrolysis, the distance between the 5′ end of the arm and the 12-kDa domains of the hexamer (R = 47 A) is the same as in the complex with the isolated ssDNA oligomer (R = 47 A) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5′ arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2–3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free Energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.
-
functional and structural heterogeneity of the dna binding site of the escherichia coli primary replicative helicase dnab protein
Journal of Biological Chemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Wlodzimierz BujalowskiAbstract:Abstract The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative Fluorescence titration, centrifugation, and Fluorescence Energy Transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude ∼10 nucleotides. ssDNA affinity for the strong subsite is ∼3 orders of magnitude higher than that for the weak subsite. Fluorescence Energy Transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5′-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.
-
complex of escherichia coli primary replicative helicase dnab protein with a replication fork recognition and structure
Biochemistry, 1998Co-Authors: Maria J. Jezewska, Surendran Rajendran, Wlodzimierz BujalowskiAbstract:Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous Fluorescence titration, Fluorescence Energy Transfer, ...
Kazuko Matsumoto - One of the best experts on this subject based on the ideXlab platform.
-
homogeneous time resolved fluoroimmunoassay of 3 5 3 triiodo l thyronine in human serum by using europium Fluorescence Energy Transfer
Talanta, 2006Co-Authors: Guilan Wang, Jingli Yuan, Kazuko MatsumotoAbstract:A sensitive homogeneous time-resolved fluoroimmunoassay method for 3,5,3'-triiodo-L-thyronine (T3) based on the Fluorescence resonance Energy Transfer (FRET) from a fluorescent Eu3+ complex, {[(4,6-dichloro-1,3,5-triazin-2-yl)amino-biphenyl-4"'-yl]-2,2':6',2"-terpyridine-6,6"-diyl}bis(methylenenitrilo) tetrakis(acetate)-Eu3+ (DTBTA-Eu3+) (lambda(ex,max)= 335 nm, lambda(em,max) = 615 nm), to an organic Fluorescence dye Cy5 has been developed. The new assay system combined the use of DTBTA-Eu3+-labeled T3-bovine serum albumin (BSA) conjugate and Cy5-labeled anti-T3 monoclonal antibody for a competitive-type immunoassay. After the competitive reactions of DTBTA-Eu3+-labeled T3-BSA and T3 sample with Cy5-labeled anti-T3 antibody, the T3 concentration was measured with a time-resolved mode by monitoring the sensitized emission of Cy5 derived from FRET in a homogeneous format. The method gives the detection limit of 0.26 ng/ml. The coefficient variations of the method are less than 2.0% and the recoveries are in the range of 80-111% for serum sample measurement. The concentrations of T3 in 30 human serum samples were determined. and the results were compared with those of the independently determined by a radio-immunoassay method. A good correlation was obtained with a correlation coefficient of 0.989. (c) 2005 Elsevier B.V. All rights reserved.
-
homogeneous time resolved fluoroimmunoassay of 3 5 3 triiodo l thyronine in human serum by using europium Fluorescence Energy Transfer
Talanta, 2006Co-Authors: Guilan Wang, Jingli Yuan, Xiaodan Hai, Kazuko MatsumotoAbstract:A sensitive homogeneous time-resolved fluoroimmunoassay method for 3,5,3'-triiodo-l-thyronine (T3) based on the Fluorescence resonance Energy Transfer (FRET) from a fluorescent Eu(3+) complex, {[(4,6-dichloro-1,3,5-triazin-2-yl)amino-biphenyl-4'''-yl]-2,2':6',2''-terpyridine-6,6''-diyl}bis(methylenenitrilo) tetrakis(acetate)-Eu(3+) (DTBTA-Eu(3+)) (lambda(ex,max)=335nm, lambda(em,max)=615nm), to an organic Fluorescence dye Cy5 has been developed. The new assay system combined the use of DTBTA-Eu(3+)-labeled T3-bovine serum albumin (BSA) conjugate and Cy5-labeled anti-T3 monoclonal antibody for a competitive-type immunoassay. After the competitive reactions of DTBTA-Eu(3+)-labeled T3-BSA and T3 sample with Cy5-labeled anti-T3 antibody, the T3 concentration was measured with a time-resolved mode by monitoring the sensitized emission of Cy5 derived from FRET in a homogeneous format. The method gives the detection limit of 0.26ng/ml. The coefficient variations of the method are less than 2.0% and the recoveries are in the range of 80-111% for serum sample measurement. The concentrations of T3 in 30 human serum samples were determined, and the results were compared with those of the independently determined by a radio-immunoassay method. A good correlation was obtained with a correlation coefficient of 0.989.
Malgorzata Maria Klonowska - One of the best experts on this subject based on the ideXlab platform.
-
close proximity of tryptophan residues and atp binding site in escherichia coli primary replicative helicase dnab protein molecular topography of the enzyme
Journal of Biological Chemistry, 1994Co-Authors: Wlodzimierz Bujalowski, Malgorzata Maria KlonowskaAbstract:Abstract The binding of fluorescent nucleotide analogs to the Escherichia coli primary replicative helicase DnaB protein causes strong quenching of protein tryptophan Fluorescence. It results from the efficient Fluorescence Energy Transfer (E) from tryptophans to analogs bound in the nucleotide-binding site, indicating that protein tryptophans are "clustered" in close proximity to the binding site. This is in contrast to the lack of detectable Energy Transfer to the fluorescent single-stranded DNA (ssDNA) derivative, suggesting a distant separation between two function-linked structural elements of the enzyme, the nucleotide- and ssDNA-binding sites. The dependence of E upon the average number of bound nucleotides/DnaB hexamer is nonlinear, implying a larger separation between tryptophans and the bound nucleotide in the low affinity sites. Spectroscopic studies reveal that tryptophan residues are located on the surface of the DnaB helicase in a hydrophobic cleft, whereas the environment of the tyrosines is heterogeneous, with 6 out of 10 tyrosine residues located on the surface of the helicase. The efficiency of the Fluorescence Energy Transfer from the tyrosines to tryptophans suggests that the "centers of mass" of the residues are separated, possibly reflecting the separation of the nucleotide- and ssDNA-binding sites, with tyrosines constituting part of the ssDNA-binding region.