Methods in molecular biology (Clifton, N.J.), 1996
Page 1. CHAPTER 12 Crystallography in the Study of Protein-DNA Interactions David G. Brown and Pa... more Page 1. CHAPTER 12 Crystallography in the Study of Protein-DNA Interactions David G. Brown and Paul S. Freemont 1. Introduction Over the past few years, there has been a dramatic increase in the num-ber of protein-DNA ...
Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in ma... more Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaP(Lm) initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaS(Lm) extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaP(Lm) and LtaS(Lm). Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaS(Lm) structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing ...
Methods in molecular biology (Clifton, N.J.), 2013
The characterization of DNA regulatory elements such as ribosome binding sites and transcriptiona... more The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ(70) transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction.
Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maint... more Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maintaining the transcriptionally repressed state of Hox/homeotic genes in a stable and heritable manner throughout development. A growing number of vertebrate genes related to the Drosophila PcG proteins have recently been identified, including two Polycomb orthologues, Pc2 and M33. PcG proteins form multiprotein complexes, termed PcG bodies, that are thought to repress transcription by altering chromatin structure. Here we report the identification and characterization of HPC3 (human Polycomb 3), a novel PcG protein isolated in a yeast two-hybrid screen using human RING1 as bait. HPC3 shows strong sequence similarity to Drosophila Pc and also to vertebrate Pc2 and M33, particularly within the chromodomain and C-box. Previous studies indicate that M33 and human Pc2 (HPC2) can interact with RING1, and we show here that HPC3 also binds to RING1. This interaction is dependent upon the HPC3 C-bo...
The human protein PML, was first identified as part of a fusion protein with retinoic acid recept... more The human protein PML, was first identified as part of a fusion protein with retinoic acid receptor alpha as found in the chromosomal translocation which gives rise to acute promyelocytic leukaemia. PML is normally localised to large matrix-associated nuclear domains (known as ND10, Kr bodies, PODS or PML NBs) which comprise several multi-protein complexes. Within the PML protein, there are a number of identified zinc-binding domains, one of which called the RING finger is found in a large family of diverse and unrelated proteins. Here, we report the effect of site-directed mutations within the context of the whole PML protein, of amino acids found on the surface of the PML RING finger domain and PML NB formation in vivo. Mutations of a small region of the RING finger domain surface affect the size and numbers of PML NBs in a mouse fibroblast expression assay, resulting in fewer but larger exogenous PML NBs. Mutations of other surface RING residues, however, do not affect exogenous ...
The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase, compris... more The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase, comprising 363 residues, was determined. The sequence was deduced by automated sequencing of CNBr-cleavage, o-iodosobenzoic acid-cleavage, trypsin-digest and staphylococcal-proteinase-digest fragments. Comparison of the sequence with other class I aldolase sequences shows that the mammalian muscle isoenzyme is one of the most highly conserved enzymes known, with only about 2% of the residues changing per 100 million years. Non-mammalian aldolases appear to be evolving at the same rate as other glycolytic enzymes, with about 4% of the residues changing per 100 million years. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Freemont (1988) Biochem. J. 249, 789-793].
The results of several secondary-structure prediction programs were combined to produce an estima... more The results of several secondary-structure prediction programs were combined to produce an estimate of the regions of alpha-helix, beta-sheet and reverse turns for fructose-bisphosphate aldolases from human and rat muscle and liver, from Trypanosoma brucei and from Drosophila melanogaster. All the aldolase sequences gave essentially the same pattern of secondary-structure predictions despite having sequences up to 50% different. One exception to this pattern was an additional strongly predicted helix in the rat liver and Drosophila enzymes. Regions of relatively high sequence variation generally were predicted as reverse turns, and probably occur as surface loops. Most of the positions corresponding to exon boundaries are located between regions predicted to have secondary-structural elements consistent with a compact structure. The predominantly alternating alpha/beta structure predicted is consistent with the alpha/beta-barrel structure indicated by preliminary high-resolution X-r...
Advances in biochemical engineering/biotechnology, 2014
As our understanding of natural biological systems grows, so too does our ability to alter and re... more As our understanding of natural biological systems grows, so too does our ability to alter and rebuild them. Synthetic biology is the application of engineering principles to biology in order to design and construct novel biological systems for specific applications. Bioluminescent organisms offer a treasure trove of light-emitting enzymes that may have applications in many areas of bioengineering, from biosensors to lighting. A few select bioluminescent organisms have been well researched and the molecular and genetic basis of their luminescent abilities elucidated, with work underway to understand the basis of luminescence in many others. Synthetic biology will aim to package these light-emitting systems as self-contained biological modules, characterize their properties, and then optimize them for use in other chassis organisms. As this catalog of biological parts grows, synthetic biologists will be able to engineer complex biological systems with the ability to emit light. These...
... in Schizosaccharomyces pombe and how the replication fork is preserved even when DNA damage i... more ... in Schizosaccharomyces pombe and how the replication fork is preserved even when DNA damage is recognized [7]. Accord-ing to Eric Enemark's (Cold Spring ... 36, 78–82 5 Bose, D., Joly, N., Pape, T., Rappas, M., Schumacher, J., Buck, M. and Zhang, X. (2008) Dissecting the ...
Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope... more Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex...
Methods in molecular biology (Clifton, N.J.), 1996
Page 1. CHAPTER 12 Crystallography in the Study of Protein-DNA Interactions David G. Brown and Pa... more Page 1. CHAPTER 12 Crystallography in the Study of Protein-DNA Interactions David G. Brown and Paul S. Freemont 1. Introduction Over the past few years, there has been a dramatic increase in the num-ber of protein-DNA ...
Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in ma... more Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaP(Lm) initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaS(Lm) extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaP(Lm) and LtaS(Lm). Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaS(Lm) structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing ...
Methods in molecular biology (Clifton, N.J.), 2013
The characterization of DNA regulatory elements such as ribosome binding sites and transcriptiona... more The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ(70) transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction.
Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maint... more Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maintaining the transcriptionally repressed state of Hox/homeotic genes in a stable and heritable manner throughout development. A growing number of vertebrate genes related to the Drosophila PcG proteins have recently been identified, including two Polycomb orthologues, Pc2 and M33. PcG proteins form multiprotein complexes, termed PcG bodies, that are thought to repress transcription by altering chromatin structure. Here we report the identification and characterization of HPC3 (human Polycomb 3), a novel PcG protein isolated in a yeast two-hybrid screen using human RING1 as bait. HPC3 shows strong sequence similarity to Drosophila Pc and also to vertebrate Pc2 and M33, particularly within the chromodomain and C-box. Previous studies indicate that M33 and human Pc2 (HPC2) can interact with RING1, and we show here that HPC3 also binds to RING1. This interaction is dependent upon the HPC3 C-bo...
The human protein PML, was first identified as part of a fusion protein with retinoic acid recept... more The human protein PML, was first identified as part of a fusion protein with retinoic acid receptor alpha as found in the chromosomal translocation which gives rise to acute promyelocytic leukaemia. PML is normally localised to large matrix-associated nuclear domains (known as ND10, Kr bodies, PODS or PML NBs) which comprise several multi-protein complexes. Within the PML protein, there are a number of identified zinc-binding domains, one of which called the RING finger is found in a large family of diverse and unrelated proteins. Here, we report the effect of site-directed mutations within the context of the whole PML protein, of amino acids found on the surface of the PML RING finger domain and PML NB formation in vivo. Mutations of a small region of the RING finger domain surface affect the size and numbers of PML NBs in a mouse fibroblast expression assay, resulting in fewer but larger exogenous PML NBs. Mutations of other surface RING residues, however, do not affect exogenous ...
The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase, compris... more The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase, comprising 363 residues, was determined. The sequence was deduced by automated sequencing of CNBr-cleavage, o-iodosobenzoic acid-cleavage, trypsin-digest and staphylococcal-proteinase-digest fragments. Comparison of the sequence with other class I aldolase sequences shows that the mammalian muscle isoenzyme is one of the most highly conserved enzymes known, with only about 2% of the residues changing per 100 million years. Non-mammalian aldolases appear to be evolving at the same rate as other glycolytic enzymes, with about 4% of the residues changing per 100 million years. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Freemont (1988) Biochem. J. 249, 789-793].
The results of several secondary-structure prediction programs were combined to produce an estima... more The results of several secondary-structure prediction programs were combined to produce an estimate of the regions of alpha-helix, beta-sheet and reverse turns for fructose-bisphosphate aldolases from human and rat muscle and liver, from Trypanosoma brucei and from Drosophila melanogaster. All the aldolase sequences gave essentially the same pattern of secondary-structure predictions despite having sequences up to 50% different. One exception to this pattern was an additional strongly predicted helix in the rat liver and Drosophila enzymes. Regions of relatively high sequence variation generally were predicted as reverse turns, and probably occur as surface loops. Most of the positions corresponding to exon boundaries are located between regions predicted to have secondary-structural elements consistent with a compact structure. The predominantly alternating alpha/beta structure predicted is consistent with the alpha/beta-barrel structure indicated by preliminary high-resolution X-r...
Advances in biochemical engineering/biotechnology, 2014
As our understanding of natural biological systems grows, so too does our ability to alter and re... more As our understanding of natural biological systems grows, so too does our ability to alter and rebuild them. Synthetic biology is the application of engineering principles to biology in order to design and construct novel biological systems for specific applications. Bioluminescent organisms offer a treasure trove of light-emitting enzymes that may have applications in many areas of bioengineering, from biosensors to lighting. A few select bioluminescent organisms have been well researched and the molecular and genetic basis of their luminescent abilities elucidated, with work underway to understand the basis of luminescence in many others. Synthetic biology will aim to package these light-emitting systems as self-contained biological modules, characterize their properties, and then optimize them for use in other chassis organisms. As this catalog of biological parts grows, synthetic biologists will be able to engineer complex biological systems with the ability to emit light. These...
... in Schizosaccharomyces pombe and how the replication fork is preserved even when DNA damage i... more ... in Schizosaccharomyces pombe and how the replication fork is preserved even when DNA damage is recognized [7]. Accord-ing to Eric Enemark's (Cold Spring ... 36, 78–82 5 Bose, D., Joly, N., Pape, T., Rappas, M., Schumacher, J., Buck, M. and Zhang, X. (2008) Dissecting the ...
Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope... more Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex...
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