The Experts below are selected from a list of 156 Experts worldwide ranked by ideXlab platform
Mitchell Lewis - One of the best experts on this subject based on the ideXlab platform.
-
functional rules for Lac Repressor operator associations and implications for protein dna interactions
Protein Science, 2010Co-Authors: Leslie Milk, Robert Daber, Mitchell LewisAbstract:The Lac Repressor has been used as a tool to understand protein–DNA recognition for many years. Recent experiments have demonstrated the ability of the Lac Repressor to control gene expression in various eukaryotic systems, making the quest for an arsenal of protein–DNA binding partners desirable for potential therapeutic applications. Here, we present the results of the most exhaustive screen of Lac Repressor-DNA binding partners to date, resulting in the elucidation of functional rules for Lac–DNA binding. Even within the confines of a single protein–DNA scaffold, modes of binding of different protein–DNA partners are sufficiently diverse so as to prevent elucidation of generalized rules for recognition for a single protein, much less an entire protein family.
-
Functional rules for Lac Repressor–operator associations and implications for protein–DNA interactions
Protein Science, 2010Co-Authors: Leslie Milk, Robert Daber, Mitchell LewisAbstract:The Lac Repressor has been used as a tool to understand protein–DNA recognition for many years. Recent experiments have demonstrated the ability of the Lac Repressor to control gene expression in various eukaryotic systems, making the quest for an arsenal of protein–DNA binding partners desirable for potential therapeutic applications. Here, we present the results of the most exhaustive screen of Lac Repressor-DNA binding partners to date, resulting in the elucidation of functional rules for Lac–DNA binding. Even within the confines of a single protein–DNA scaffold, modes of binding of different protein–DNA partners are sufficiently diverse so as to prevent elucidation of generalized rules for recognition for a single protein, much less an entire protein family.
-
the Lac Repressor
Comptes Rendus Biologies, 2005Co-Authors: Mitchell LewisAbstract:Abstract Few proteins have had such a strong impact on a field as the Lac Repressor has had in Molecular Biology. Over 40 years ago, Jacob and Monod [Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3 (1961) 318] proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. It is a cogent depiction of how a set of ‘structural’ genes may be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. In bacteria, the genes required for Lactose utilization are negatively regulated when a Repressor molecule binds to an upstream cis activated operator. The Repressor and its operator together form a genetic switch, the Lac operon. The switch functions when inducer molecules alter the conformation of the Repressor in a specific manner. In the presence of a particular metabolite, the Repressor undergoes a conformational change that reduces its affinity for the operator. The structures of the Lac Repressor and its complexes with operator DNA and effector molecules have provided a physical platform for visualizing at the molecular level the different conformations the Repressor and the molecular basis for the switch. The structures of Lac Repressor, bound to its operator and inducer, have also been invaluable for interpreting a plethora of biochemical and genetic data. To cite this article: M. Lewis, C. R. Biologies 328 (2005).
-
a closer view of the conformation of the Lac Repressor bound to operator
Nature Structural & Molecular Biology, 2000Co-Authors: Charles E Bell, Mitchell LewisAbstract:Crystal structures of the Lac Repressor, with and without isopropylthiogaLactoside (IPTG), and the Repressor bound to operator have provided a model for how the binding of the inducer reduces the affinity of the Repressor for the operator. However, because of the low resolution of the operator-bound structure (4.8 A), the model for the allosteric transition was presented in terms of structural elements rather than in terms of side chain interactions. Here we have constructed a dimeric Lac Repressor and determined its structure at 2.6 A resolution in complex with a symmetric operator and the anti-inducer orthonitrophenylfucoside (ONPF). The structure enables the induced (IPTG-bound) and repressed (operator-bound) conformations of the Repressor to be compared in atomic detail. An extensive network of interactions between the DNA-binding and core domains of the Repressor suggests a possible mechanism for the allosteric transition.
-
Lac Repressor genetic map in real space
Trends in Biochemical Sciences, 1997Co-Authors: H C Pace, Jeffrey H Miller, M A Kercher, Ponzy Lu, Peter Markiewicz, Geoffrey Chang, Mitchell LewisAbstract:Abstract Here, we present a graphic display of the phenotypes of more than 4000 single amino acid substitution mutations on the three-dimensional structure of the Lac Repressor tetramer bound to DNA. The genetic data and the X-ray diffraction studies contribute to define an allosteric mechanism and yield a visual demonstration of the importance of core or buried residues in protein structure.
Rianne Kaptein - One of the best experts on this subject based on the ideXlab platform.
-
novel strategies to overcome expression problems encountered with toxic proteins application to the production of Lac Repressor proteins for nmr studies
Protein Expression and Purification, 2009Co-Authors: Julija Romanuka, Heidi Van Den Bulke, Rianne Kaptein, Rolf Boelens, Gert E. FolkersAbstract:Abstract NMR studies of structural aspects of allosteric regulation by the Lac Repressor requires overexpression and isotope labeling of the protein. The size of the Repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac Repressor and its core domain mutants. The Lac Repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac Repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac Repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D 2 O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.
-
plasticity in protein dna recognition Lac Repressor interacts with its natural operator o1 through alternative conformations of its dna binding domain
The EMBO Journal, 2002Co-Authors: Charalampos G Kalodimos, Rolf Boelens, Alexandre M J J Bonvin, Roberto K Salinas, Rainer Wechselberger, Rianne KapteinAbstract:The Lac Repressor–operator system is a model system for understanding protein–DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both Repressor and operator, the specific recognition mechanism of the natural Lac operators by Lac Repressor has remained elusive. Here we present the first high-resolution structure of a dimer of the DNA-binding domain of Lac Repressor bound to its natural operator O1. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48° of the right Lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the Lac Repressor–operator system is unique among other protein–DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.
-
hinge helix formation and dna bending in various Lac Repressor operator complexes
The EMBO Journal, 1999Co-Authors: Christian A E M Spronk, Gert E. Folkers, Rolf Boelens, Rainer Wechselberger, Annemarie Noordman, Nienke Van Den Brink, Rianne KapteinAbstract:The hinge-region of the Lac Repressor plays an important role in the models for induction and DNA looping in the Lac operon. When Lac Repressor is bound to a tight-binding symmetric operator, this region forms an alpha-helix that induces bending of the operator. The presence of the hinge-helices is questioned by previous data that suggest that the Repressor does not bend the wild-type operator. We show that in the wild-type complex the hinge-helices are formed and the DNA is bent, similar to the symmetric complex. Furthermore, our data show differences in the binding of the DNA binding domains to the half-sites of the wild-type operator and reveal the role of the central base-pair of the wild-type operator in the Repressor-operator interaction. The differences in binding to the operator half-sites are incorporated into a model that explains the relative affinities of the Repressor for various Lac operator sequences that contain left and right half-sites with different spacer lengths.
-
the solution structure of Lac Repressor headpiece 62 complexed to a symmetrical Lac operator
Structure, 1999Co-Authors: Christian A E M Spronk, Rolf Boelens, Alexandre M J J Bonvin, Plachikkat K Radha, Giuseppe Melacini, Rianne KapteinAbstract:Abstract Background: Lactose Repressor protein (Lac) controls the expression of the Lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the Lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein–protein and protein–DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail. Results: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac Repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized Lac operator. Analysis of the structures reveals specific interactions between Lac Repressor and DNA that were not found in previously investigated Lac Repressor–DNA complexes. Important differences with the previously reported structures of the HP56–DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein–protein and protein–DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur Repressor–DNA complexes. Conclusions: The structures of the HP62–DNA complex provide the basis for a better understanding of the specific recognition in the Lac Repressor–operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein–protein and protein–DNA interactions responsible for the high affinity of the Repressor for operator DNA.
Ponzy Lu - One of the best experts on this subject based on the ideXlab platform.
-
Lac Repressor genetic map in real space
Trends in Biochemical Sciences, 1997Co-Authors: H C Pace, Jeffrey H Miller, M A Kercher, Ponzy Lu, Peter Markiewicz, Geoffrey Chang, Mitchell LewisAbstract:Abstract Here, we present a graphic display of the phenotypes of more than 4000 single amino acid substitution mutations on the three-dimensional structure of the Lac Repressor tetramer bound to DNA. The genetic data and the X-ray diffraction studies contribute to define an allosteric mechanism and yield a visual demonstration of the importance of core or buried residues in protein structure.
-
Lac Repressor operator complex
Current Opinion in Structural Biology, 1997Co-Authors: M A Kercher, Ponzy Lu, Mitchell LewisAbstract:Abstract For many years the Lac operon of Escherichia coli has been the paradigm for gene regulation. Recently, the structures of the Lac Repressor core bound to isopropyl-β- D -1-thiogaLactoside (IPTG), the intact apo Lac Repressor, the intact Lac Repressor complexes with IPTG and a 21-base-pair symmetric operator, and the refined headpiece of the Repressor have been determined. These structures have provided a framework for understanding a wealth of biochemical and genetic information. An analysis of these structures, as well as a description of their function and a comparison to homologous proteins, is now possible.
-
Escherichia coli Lac Repressor-Lac operator interaction and the influence of allosteric effectors.
Journal of Molecular Biology, 1997Co-Authors: Nancy C. Horton, Mitchell Lewis, Ponzy LuAbstract:Abstract The wild type E. coli Lac operator is embedded in a 35 base-pair DNA sequence containing extensive 2-fold symmetry, suggesting a symmetric Repressor operator complex. However, deviations from strict 2-fold symmetry occur at the central base-pair and at three additional base-pairs. Using an operator fragment binding analysis we have determined: (a) a relative contribution each pair provides to the Lac Repressor- Lac operator DNA complex, (b) the operator DNA length necessary for maximum binding to Lac Repressor; and (c) the contribution of the several non-symmetric base in the wild-type operator to the binding affinity. Since Lac Repressor- Lac operator DNA interaction is reduced upon binding of the gratuitous inducer, isopropyl-β-D-gaLactoside (IPTG), the same DNA fragment binding analysis was performed with the low affinity form of Lac Repressor. In the presence of inducer, the affinity for the left half site of the wild-type Lac operator is reduced without significant reduction on the right half of the operator. Conversely, the anti-inducer orthonitrophenylfucoside (ONPF) which stabilizes the Lac Repressor- Lac operator complex increases the binding affinity, particularly to the right half of the operator.
-
In vivo interaction of Escherichia coli Lac Repressor N-terminal fragments with the Lac operator☆
Journal of Molecular Biology, 1991Co-Authors: Anastasia M. Khoury, Harry S. Nick, Ponzy LuAbstract:Abstract Escherichia coli Lac Repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with pro teases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo , the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/ promoter. In addition to the wild-type LacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3 → Tyr) and 61 (Ser61 → Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact Lac Repressor by a factor of 10 2 to 10 4 . The expression of these Lac Repressor fragments in the cell was verified by radioimmuno-assays. Both wild-type and mutant Lac Repressor N termini bound operator DNA as judged by reduced β-gaLactosidase synthesis and methylation protection in vivo . These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.
Rolf Boelens - One of the best experts on this subject based on the ideXlab platform.
-
novel strategies to overcome expression problems encountered with toxic proteins application to the production of Lac Repressor proteins for nmr studies
Protein Expression and Purification, 2009Co-Authors: Julija Romanuka, Heidi Van Den Bulke, Rianne Kaptein, Rolf Boelens, Gert E. FolkersAbstract:Abstract NMR studies of structural aspects of allosteric regulation by the Lac Repressor requires overexpression and isotope labeling of the protein. The size of the Repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac Repressor and its core domain mutants. The Lac Repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac Repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac Repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D 2 O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.
-
plasticity in protein dna recognition Lac Repressor interacts with its natural operator o1 through alternative conformations of its dna binding domain
The EMBO Journal, 2002Co-Authors: Charalampos G Kalodimos, Rolf Boelens, Alexandre M J J Bonvin, Roberto K Salinas, Rainer Wechselberger, Rianne KapteinAbstract:The Lac Repressor–operator system is a model system for understanding protein–DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both Repressor and operator, the specific recognition mechanism of the natural Lac operators by Lac Repressor has remained elusive. Here we present the first high-resolution structure of a dimer of the DNA-binding domain of Lac Repressor bound to its natural operator O1. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48° of the right Lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the Lac Repressor–operator system is unique among other protein–DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.
-
hinge helix formation and dna bending in various Lac Repressor operator complexes
The EMBO Journal, 1999Co-Authors: Christian A E M Spronk, Gert E. Folkers, Rolf Boelens, Rainer Wechselberger, Annemarie Noordman, Nienke Van Den Brink, Rianne KapteinAbstract:The hinge-region of the Lac Repressor plays an important role in the models for induction and DNA looping in the Lac operon. When Lac Repressor is bound to a tight-binding symmetric operator, this region forms an alpha-helix that induces bending of the operator. The presence of the hinge-helices is questioned by previous data that suggest that the Repressor does not bend the wild-type operator. We show that in the wild-type complex the hinge-helices are formed and the DNA is bent, similar to the symmetric complex. Furthermore, our data show differences in the binding of the DNA binding domains to the half-sites of the wild-type operator and reveal the role of the central base-pair of the wild-type operator in the Repressor-operator interaction. The differences in binding to the operator half-sites are incorporated into a model that explains the relative affinities of the Repressor for various Lac operator sequences that contain left and right half-sites with different spacer lengths.
-
the solution structure of Lac Repressor headpiece 62 complexed to a symmetrical Lac operator
Structure, 1999Co-Authors: Christian A E M Spronk, Rolf Boelens, Alexandre M J J Bonvin, Plachikkat K Radha, Giuseppe Melacini, Rianne KapteinAbstract:Abstract Background: Lactose Repressor protein (Lac) controls the expression of the Lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the Lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein–protein and protein–DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail. Results: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac Repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized Lac operator. Analysis of the structures reveals specific interactions between Lac Repressor and DNA that were not found in previously investigated Lac Repressor–DNA complexes. Important differences with the previously reported structures of the HP56–DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein–protein and protein–DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur Repressor–DNA complexes. Conclusions: The structures of the HP62–DNA complex provide the basis for a better understanding of the specific recognition in the Lac Repressor–operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein–protein and protein–DNA interactions responsible for the high affinity of the Repressor for operator DNA.
M A Kercher - One of the best experts on this subject based on the ideXlab platform.
-
Lac Repressor genetic map in real space
Trends in Biochemical Sciences, 1997Co-Authors: H C Pace, Jeffrey H Miller, M A Kercher, Ponzy Lu, Peter Markiewicz, Geoffrey Chang, Mitchell LewisAbstract:Abstract Here, we present a graphic display of the phenotypes of more than 4000 single amino acid substitution mutations on the three-dimensional structure of the Lac Repressor tetramer bound to DNA. The genetic data and the X-ray diffraction studies contribute to define an allosteric mechanism and yield a visual demonstration of the importance of core or buried residues in protein structure.
-
Lac Repressor operator complex
Current Opinion in Structural Biology, 1997Co-Authors: M A Kercher, Ponzy Lu, Mitchell LewisAbstract:Abstract For many years the Lac operon of Escherichia coli has been the paradigm for gene regulation. Recently, the structures of the Lac Repressor core bound to isopropyl-β- D -1-thiogaLactoside (IPTG), the intact apo Lac Repressor, the intact Lac Repressor complexes with IPTG and a 21-base-pair symmetric operator, and the refined headpiece of the Repressor have been determined. These structures have provided a framework for understanding a wealth of biochemical and genetic information. An analysis of these structures, as well as a description of their function and a comparison to homologous proteins, is now possible.
