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Research

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Dr Ehmke Pohl

Telephone: +44 (0) 191 33 43619
Member of the Durham X-ray Centre
Telephone: +44 (0) 191 33 43619

(email at ehmke.pohl@durham.ac.uk)

Research Interest

The overall goal of the group is to determine the 3-D structures of proteins with relevance to biomedicine or biotechnology. Our primary technique is protein crystallography complemented by a wide range of biophysical techniques ranging from small angle X-ray scattering and electron microscopy to isothermal titration calorimetry and thermal shift assays.

Virus-X: Viral Metagenomics for Innovation value

virus-x

In this EU Horizon2020 funded project a consortium of private companies, led by the Icelandic biotech company Prokazyme (http://prokazyme.com/) and public researchers. will mine the unexplored genomic diversity if bacteriophages from extreme environments. Starting from a massive Next-generation sequencing effort, the goal is to discover new enzymes with novel properties for biotechnological applications. Our group is responsible for the biophysical and structural characterisation of proteins with a specific focus on DNA/RNA binding and processing enzymes.

Structure and mechanism of transcriptional regulator proteins

The viability of all bacteria depends on their quick adaptation to the ever-changing environment. In many cases, gene-expression in response to internal and/or external signals is controlled by transcriptional regulators. Using a combination of crystallographic, biophysical and computational tools we investigate ligand binding and the mechanism of DNA recognition. We have solved a number of crystal structures of the Catabolite Activator Protein (CAP) from E. coli and its closely related homologue GlxR from C. glutamicum to dissect structural and dynamic contributions of allosteric ligand and DNA binding. (Rodger, T.L. et al. Plos Biol (2013) e1001651; Townsend, P.D. PLoSOne (2014) e113265).

Superposition of crystal structures of Apo GlxR (red) with cAMP bound GlxR (orange) showing the motion of DNA-binding helices.

Superposition of crystal structures of Apo GlxR (red) with cAMP bound GlxR (orange) showing the motion of DNA-binding helices.

Thermal Shift Assays for protein crystallography

Thermal Shift Assays (TSA) also known as Differential Scanning Fluorimetry or Thermofluor assays are based on the change of fluorescence signal of a chemical probe. This assay can easily be performed using SYPRO orange or a related dye and a standard quantitative RT-PCR instrument. Using SBS 96-well plates the protein-dye mix is slowly heated while the fluorescence signal is monitored. When the protein chain unfolds the hydrophobic core becomes exposed and the fluorescence signal increases until complete denaturation. We have developed new screens in 96-well plate format to investigate protein stability and aid crystallization efforts. Data analysis and interpretation is automatically done by a freely available program NAMI, which reads the raw data, calculates melting temperature and displays the results in the form of color-coded table (Groftehauge, M.K., Hajizadeh, N.R., Swann, M.J., Pohl, E., (2015) Acat Cryst D71:36-44).

Experimental fluorescence data from one representative well (blue dots), first derivative and calculated Tm of 66 ˚C.

Experimental fluorescence data from one representative well (blue dots), first derivative and calculated Tm of 66 ˚C.

Results from the Durham salt screen for crystallization. Blue indicates a significant increase in Tm (stabilization), red a decrease.

Results from the Durham salt screen for crystallization. Blue indicates a significant increase in Tm (stabilization), red a decrease.

The program NAMI can be downloaded here, and the Durham screens will soon be available via Molecular Dimensions (http://www.moleculardimensions.com/)

Miscellaneous projects

Additional projects in the group are focused on the structure-based design of new ligands, probes and inhibitor inlcuding

  • Investigating the structure and function of proteins involved in the sphingolipid biosynthesis from pathogenic protozoa in collaboration with Dr. P.W. Denny (Durham)
  • Design of new chemical probes targeting the Retionic Acid Receptors (RAR) proteins with Prof. A. Whiting (Durham) and High Force Research (http://www.highforceresearch.com/)
  • Structure-based design of Tuberculosis booster drugs in collaboration with the Dr. JC Cole form Cambridge Crystallographic Data Center (http://www.ccdc.cam.ac.uk/) and Dr. A.R. Baulard, Pasteur Institute, Lille.
  • Development of new inhibitors for Multi-herbicide resistant weeds in collaboration with Professor P.G. Steel (Durham), Prof. R. Edwards (Newcastle) and Syngenta (https://www.syngenta.co.uk/)

Protein Crystallography Laboratory

Our group is located in the joined Biophysical Sciences Institute (BSI) laboratories in the Chemistry Department of Durham University. The protein crystallography laboratory has full access to state-of the art protein production and purification equipment (Harbiger Bioreactor, AKTA Explorer, AKTA Pure), biophysical characterization (CD, TSA, FA, ITC, SPR and MS) and crystal structure determination (Innovadyne Screenmaker, Bruker MicroStar, Bruker D8).

Acknowledgments

Financial support has been or is currently generously provided by the EPSRC, BBSRC, MRC, The Royal Society, the Wellcome Trust, the British Society for Antimicrobial Chemotherapy and Horizon2020.

Research Groups

Department of Biosciences

Department of Chemistry

Wolfson Research Institute for Health and Wellbeing

Research Interests

  • Protein Crystallography
  • DNA-Binding Proteins

Publications

Journal Article

Supervises