Imperial College London

ProfessorPaulFreemont

Faculty of MedicineDepartment of Infectious Disease

Chair in Protein Crystallography
 
 
 
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Contact

 

+44 (0)20 7594 5327p.freemont

 
 
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Location

 

259Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

255 results found

Webb AJ, Kelwick R, Freemont PS, 2017, Opportunities for applying whole-cell bioreporters towards parasite detection, Microbial Biotechnology, Vol: 10, Pages: 244-249, ISSN: 1751-7915

Journal article

Hazel P, Kroll SH, Bondke A, Barbazanges M, Patel H, Fuchter MJ, Coombes RC, Ali S, Barrett AG, Freemont PSet al., 2017, Inhibitor selectivity for cyclin-dependent kinase 7: a structural, thermodynamic, and modelling study, Chemmedchem, Vol: 12, Pages: 372-380, ISSN: 1860-7187

Deregulation of the cell cycle by mechanisms that lead to elevated activities of cyclin-dependent kinases (CDK) is a feature of many human diseases, cancer in particular. We identified small-molecule inhibitors that selectively inhibit CDK7, the kinase that phosphorylates cell-cycle CDKs to promote their activities. To investigate the selectivity of these inhibitors we used a combination of structural, biophysical, and modelling approaches. We determined the crystal structures of the CDK7-selective compounds ICEC0942 and ICEC0943 bound to CDK2, and used these to build models of inhibitor binding to CDK7. Molecular dynamics (MD) simulations of inhibitors bound to CDK2 and CDK7 generated possible models of inhibitor binding. To experimentally validate these models, we gathered isothermal titration calorimetry (ITC) binding data for recombinant wild-type and binding site mutants of CDK7 and CDK2. We identified specific residues of CDK7, notably Asp155, that are involved in determining inhibitor selectivity. Our MD simulations also show that the flexibility of the G-rich and activation loops of CDK7 is likely an important determinant of inhibitor specificity similar to CDK2.

Journal article

Moore SJ, lai H-E, Kelwick R, Mei S, Bell DJ, Polizzi K, Freemont PSet al., 2016, EcoFlex - a multifunctional MoClo kit for E. coli synthetic biology, ACS Synthetic Biology, Vol: 5, Pages: 1059-1069, ISSN: 2161-5063

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology.

Journal article

MacDonald J, Freemont PS, 2016, Computational protein design with backbone plasticity, Biochemical Society Transactions, Vol: 44, Pages: 1523-1529, ISSN: 1470-8752

The computational algorithms used in the design of artificial proteins have become increasingly sophisticated in recent years, producing a series of remarkable successes. The most dramatic of these is the de novo design of artificial enzymes. The majority of these designs have reused naturally occurring protein structures as ‘scaffolds’ onto which novel functionality can be grafted without having to redesign the backbone structure. The incorporation of backbone flexibility into protein design is a much more computationally challenging problem due to the greatly increased search space, but promises to remove the limitations of reusing natural protein scaffolds. In this review, we outline the principles of computational protein design methods and discuss recent efforts to consider backbone plasticity in the design process.

Journal article

Kelwick RJR, Webb AJ, MacDonald JT, Freemont PSet al., 2016, Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements, Metabolic Engineering, Vol: 38, Pages: 370-381, ISSN: 1096-7184

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8 µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.

Journal article

MacDonald JT, Kabasakal BV, Godding D, Kraatz S, Henderson L, Barber J, Freemont PS, Murray JWet al., 2016, Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: 10346-10351, ISSN: 1091-6490

The ability to design and construct structures with atomic level precisionis one of the key goals of nanotechnology. Proteins offer anattractive target for atomic design, as they can be synthesized chemicallyor biologically, and can self-assemble. However the generalizedprotein folding and design problem is unsolved. One approach tosimplifying the problem is to use a repetitive protein as a scaffold.Repeat proteins are intrinsically modular, and their folding and structuresare better understood than large globular domains. Here, wehave developed a new class of synthetic repeat protein, based onthe pentapeptide repeat family of beta-solenoid proteins. We haveconstructed length variants of the basic scaffold, and computationallydesigned de novo loops projecting from the scaffold core. Theexperimentally solved 3.56 ˚A resolution crystal structure of one designedloop matches closely the designed hairpin structure, showingthe computational design of a backbone extension onto a syntheticprotein core without the use of backbone fragments from knownstructures. Two other loop designs were not clearly resolved in thecrystal structures and one loop appeared to be in an incorrect conformation.We have also shown that the repeat unit can accommodatewhole domain insertions by inserting a domain into one of the designedloops.

Journal article

Schuster C, Bellows L, Tosi T, Campeotto, Corrigan, Freemont P, Grundling Aet al., 2016, The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus, Science Signaling, Vol: 9, Pages: ra81-ra81, ISSN: 1945-0877

Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. To survive an increase in osmolarity, bacteria immediately take up potassium ions and small organic compounds known as compatible solutes. The second messenger cyclic diadenosine monophosphate (c-di-AMP) reduces the ability of bacteria to withstand osmotic stress by binding to and inhibiting several proteins that promote potassium uptake. We identified OpuCA, the adenosine triphosphatase (ATPase) component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S. aureus and found that the LAC*ΔgdpP strain of S. aureus, which overproduces c-di-AMP, showed reduced carnitine uptake. The paired cystathionine-β-synthase (CBS) domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP inhibits osmoprotection through multiple mechanisms.

Journal article

Filloux A, Freemont P, 2016, Structural biology: baseplates in contractile machines, Nature Microbiology, Vol: 1, ISSN: 2058-5276

Journal article

Chambers S, Kitney R, Freemont P, 2016, The Foundry: the DNA synthesis and construction Foundry at Imperial College., Biochemical Society Transactions, Vol: 44, Pages: 687-688, ISSN: 1470-8752

The establishment of a DNA synthesis and construction foundry at Imperial College in London heralds a new chapter in the development of synthetic biology to meet new global challenges. The Foundry employs the latest technology to make the process of engineering biology easier, faster and scalable. The integration of advanced software, automation and analytics allows the rapid design, build and testing of engineered organisms.

Journal article

Planamente S, Salih O, Manoli E, Albesa-Jove D, Freemont PS, Filloux AAMet al., 2016, TssA forms a gp6-like ring attached to the type VI secretion sheath, EMBO Journal, Vol: 35, Pages: 1613-1627, ISSN: 0261-4189

The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo. In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS. Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS.

Journal article

Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, de Arroyo Garcia L, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont P, Kitney RI, Reeve B, Ellis Tet al., 2016, Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E3431-E3440, ISSN: 1091-6490

Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae. Its high strength, purity and biocompatibility make it of great interest to materials science, however precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a new strain of Komagataeibacter rhaeticus (Komagataeibacter rhaeticus iGEM) that can produce cellulose at high yields, grow in low nitrogen conditions, and is highly resistant to toxic chemicals. We achieve external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering and biotechnology.

Journal article

Lund-Palau H, Turnbull AR, Bush A, Bardin E, Cameron L, Soren O, Wierre-Gore N, Alton EW, Bundy JG, Connett G, Faust SN, Filloux A, Freemont P, Jones A, Khoo V, Morales S, Murphy R, Pabary R, Simbo A, Schelenz S, Takats Z, Webb J, Williams HD, Davies JCet al., 2016, Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches, Expert Review of Respiratory Medicine, Vol: 10, Pages: 685-697, ISSN: 1747-6348

Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development.

Journal article

Webb AJ, Kelwick R, Doenhoff MJ, Kylilis N, MacDonald J, Wen KY, Mckeown C, Baldwin G, Ellis T, Jensen K, Freemont PSet al., 2016, A protease-based biosensor for the detection of schistosome cercariae, Scientific Reports, Vol: 6, ISSN: 2045-2322

Parasitic diseases affect millions of people worldwide, causing debilitating illnesses anddeath. Rapid and cost-effective approaches to detect parasites are needed, especially inresource-limited settings. A common signature of parasitic diseases is the release of specificproteases by the parasites at multiple stages during their life cycles. To this end, weengineered several modular Escherichia coli and Bacillus subtilis whole-cell-basedbiosensors which incorporate an interchangeable protease recognition motif into theirdesigns. Herein, we describe how several of our engineered biosensors have been applied todetect the presence and activity of elastase, an enzyme released by the cercarial larvae stageof Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes areresponsible for the infection of an estimated 200 million people worldwide. Since ourbiosensors are maintained in lyophilised cells, they could be applied for the detection of S.mansoni and other parasites in settings without reliable cold chain access.

Journal article

Florea M, Reeve B, Abbott J, Freemont PS, Ellis Tet al., 2016, Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582., Scientific Reports, Vol: 6, ISSN: 2045-2322

Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.

Journal article

Yu N, Nützmann HW, MacDonald JT, Moore B, Field B, Berriri S, Trick M, Rosser SJ, Kumar SV, Freemont PS, Osbourn Aet al., 2016, Delineation of metabolic gene clusters in plant genomes by chromatin signatures., Nucleic Acids Research, Vol: 44, Pages: 2255-2265, ISSN: 1362-4962

Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.

Journal article

Kelwick R, Webb AJ, Macdonald JT, Freemont PSet al., 2016, Development of a bacillus subtilis cell-free transcriptiontranslation system

Conference paper

Kopniczky M, Jensen K, Freemont P, 2016, Introducing the human cell-free TX-TL system as a new prototyping platform for mammalian synthetic biology

Conference paper

Polizzi KM, Freemont PS, 2016, Synthetic biology biosensors for healthcare and industrial biotechnology applications

Conference paper

Trusch F, Matena A, Vuk M, Koerver L, Knaevelsrud H, Freemont PS, Meyer H, Bayer Pet al., 2015, The N-terminal region of the ubiquitin regulatory x (UBX) domain-containing Protein 1 (UBXD1) modulates interdomain communication within the valosin-containing Protein p97, Journal of Biological Chemistry, Vol: 290, Pages: 29414-29427, ISSN: 1083-351X

Journal article

Kopniczky M, moore S, freemont P, 2015, Multilevel regulation and translational switches in synthetic biology, IEEE Transactions on Biomedical Circuits and Systems, Vol: 9, Pages: 485-496, ISSN: 1940-9990

In contrast to the versatility of regulatory mechanisms in natural systems, synthetic genetic circuits have been so far predominantly composed of transcriptionally regulated modules. This is about to change as the repertoire of foundational tools for post-transcriptional regulation is quickly expanding. We provide an overview of the different types of translational regulators: protein, small molecule and RNA responsive and we describe the new emerging circuit designs utilizing these tools. There are several advantages of achieving multilevel regulation via translational switches and it is likely that such designs will have the greatest and earliest impact in mammalian synthetic biology for regenerative medicine and gene therapy applications.

Journal article

Kopniczky M, freemont P, moore S, Multilevel regulation and translational switches in synthetic biology, IEEE Transactions on Biomedical Circuits and Systems, ISSN: 1940-9990

Journal article

Kopniczky M, freemont P, Moore S, Multilevel regulation and translational switches in synthetic biology, IEEE Transactions on Biomedical Circuits and Systems, ISSN: 1940-9990

In contrast to the versatility of regulatory mechanisms in natural systems, synthetic genetic circuits have been so far predominantly composed of transcriptionally regulated modules. This is about to change as the repertoire of foundational tools for post-transcriptional regulation is quickly expanding. We provide an overview of the different types of translational regulators: protein, small molecule and RNA responsive and we describe the new emerging circuit designs utilizing these tools. There are several advantages of achieving multilevel regulation via translational switches and it is likely that such designs will have the greatest and earliest impact in mammalian synthetic biology for regenerative medicine and gene therapy applications.

Journal article

Weston DJ, Russell RA, Batty E, Jensen K, Stephens DA, Adams NM, Freemont PSet al., 2015, New quantitative approaches reveal the spatial preference of nuclear compartments in mammalian fibroblasts, JOURNAL OF THE ROYAL SOCIETY INTERFACE, Vol: 12, ISSN: 1742-5689

Journal article

Kelwick R, Kopniczky M, Bower I, Chi W, Chin MHW, Fan S, Pilcher J, Strutt J, Webb AJ, Jensen K, Stan G-B, Kitney R, Freemont Pet al., 2015, A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli, PLOS ONE, Vol: 10, ISSN: 1932-6203

Journal article

Campeotto I, Zhang Y, Mladenov MG, Freemont PS, Grundling Aet al., 2015, Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA, the Founding Member of a New Signal Transduction Protein Family, Journal of Biological Chemistry, Vol: 290, Pages: 2888-2901, ISSN: 1083-351X

Signaling nucleotides are integral parts of signal transductionsystems allowing bacteria to cope with and rapidly respond tochanges in the environment. The Staphylococcus aureus PII-likesignal transduction protein PstA was recently identified as acyclic diadenylate monophosphate (c-di-AMP)-binding protein.Here, we present the crystal structures of the apo- and c-diAMP-boundPstA protein, which is trimeric in solution as wellas in the crystals. The structures combined with detailed bioinformaticsanalysis revealed that the protein belongs to a newfamily of proteins with a similar core fold but with distinct featuresto classical PII proteins, which usually function in nitrogenmetabolism pathways in bacteria. The complex structurerevealed three identical c-di-AMP-binding sites per trimer witheach binding site at a monomer-monomer interface. Althoughdistinctly different from other cyclic-di-nucleotide-bindingsites, as the half-binding sites are not symmetrical, the complexstructure also highlighted common features for c-di-AMPbindingsites. A comparison between the apo and complexstructures revealed a series of conformational changes thatresult in the ordering of two anti-parallel !-strands that protrudefrom each monomer and allowed us to propose a mechanismon how the PstA protein functions as a signaling transductionprotein.

Journal article

Kelwick R, MacDonald JT, Webb AJ, Freemont Pet al., 2014, Developments in the Tools and Methodologies of Synthetic Biology, Frontiers in Bioengineering and Biotechnology, Vol: 2

Journal article

Foerster A, Planamente S, Manoli E, Lossi NS, Freemont PS, Filloux Aet al., 2014, Coevolution of the ATPase ClpV, the Sheath Proteins TssB and TssC, and the Accessory Protein TagJ/HsiE1 Distinguishes Type VI Secretion Classes, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289

Journal article

Campeotto I, Percy MG, MacDonald JT, Foerster A, Freemont PS, Gruendling Aet al., 2014, Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System, Journal of Biological Chemistry, Vol: 289, Pages: 28054-28069, ISSN: 0021-9258

Lipoteichoic acid (LTA) is an important cell wall componentrequired for proper cell growth in many Gram-positive bacteria.In Listeria monocytogenes, two enzymes are required for the synthesisof this polyglycerolphosphate polymer. The LTA primaseLtaPLm initiates LTA synthesis by transferring the first glycerolphosphate(GroP) subunit onto the glycolipid anchor and theLTA synthase LtaSLm extends the polymer by the repeated additionof GroP subunits to the tip of the growing chain. Here, wepresent the crystal structures of the enzymatic domains ofLtaPLm and LtaSLm. Although the enzymes share the same fold,substantial differences in the cavity of the catalytic site andsurface charge distribution contribute to enzyme specialization.The eLtaSLm structure was also determined in complexwith GroP revealing a second GroP binding site. Mutationalanalysis confirmed an essential function for this binding siteand allowed us to propose a model for the binding of thegrowing chain.

Journal article

Polizzi KM, Kylilis N, Lai HE, Freemont PSet al., 2014, Detecting protein biomarkers using engineered biosensors based on synthetic biology principles, 248th National Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727

Conference paper

Casini A, Christodoulou G, Freemont PS, Baldwin GS, Ellis T, MacDonald JTet al., 2014, R2oDNA Designer: Computational Design of Biologically Neutral Synthetic DNA Sequences, ACS SYNTHETIC BIOLOGY, Vol: 3, Pages: 525-528, ISSN: 2161-5063

Journal article

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