Investigation of NisK/NisR

 

Lab rotation project description

This will provide exposure to and training in a widely used molecular docking programme, AutoDock, and will introduce related biomolecular systems of interest. Well-developed tutorials in both AutoDock and Unix will be completed in the first week. The tasks to complete are as follows:

  • weeks two and three: rigid body docking of truncated nisin and pyrophosphate; exploration of parameter settings in AutoDock; understanding, processing and visualisation of results.
  • weeks four and five: docking of truncated nisin to flexible pyrophosphate; characterisation and analysis of complexes
  • week six: docking of flexible nisin to rigid pyrophosphate; identification of key degrees of freedom; application of clustering algorithms to analyse structural variation and commonalities.

The objective is to give in a nutshell a flavour of the continuity in biological research from design and sample preparation through analysis to functional characterisation. The rotation will be delivered as follows:

  • weeks one and two: antimicrobial peptide nisin, E234, will be produced and purified
  • weeks three and four: model lipid membranes containing a molecular partner for nisin will be prepared and antibiotic/target interactions within lipid membranes will be studied by solid state NMR
  • weeks five, six and seven: Functional characterisation will be carried out using fluorescence dequenching assays

Fact file

Research theme

MCO

Location

TBC

Rotation

TBC

Contact

2nd supervisor

Boyan Bonev 

BBSRC Doctoral Training Partnerships
 

Linked PhD Project Outline

Nisin, E234, is an efficient and safe antimicrobial peptide with target-selective action, used worldwide as a food preservative. Resistance and production of nisin in L. lactis is autoregulated via the NisK/R two-component system through a quorum sensing mechanism. NisK is a histidine sensor kinase that is localized in the cytoplasmic membrane and serves as a receptor for nisin. Extracellular nisin activates surface receptor NisK and initiates a signal transduction cascade, which begins with autophosphorylation of NisK. Phosphorylated NisK transfers a phosphoryl group to NisR, which recruits downstream response regulators to nisA and nisF operators, the nisin biosynthesis genes. We seek structural characterisation of the receptor and activated complex in wild type and constitutively phosphorylated engineered NisK alone and in activated complexes with nisin and with downstream signal transducer, NisR. This will be done using advanced computational techniques, NMR, crystallography and circular dichroism. 

The project builds on our expertise in lantionine antimicrobials and molecular engineering. The project also aims to engineer nisin-informed constitutive activators of the receptor system for increased antimicrobial production and explore their role as decoys in adjuvant antimicrobial therapies.

 

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946
Email: bbdtp@nottingham.ac.uk