Summary

FtsZ is a prokaryotic homologue of tubulin which forms a band, the z-ring, around the cell wall during cell division at the location where the cell will divide. It is the foundation of the molecular machine that causes cell wall invagination and septation. The Z-ring is constructed from meta-stable FtsZ protofilaments which are stabilised by ligands such as GTP. The way in which protofilament stabilisation occurs and the kinetics of this process can be investigated using Linear Dichroism (LD) and 90° Light Scattering.

In this mini project I did further development work on a new LD cell based on a stationary capillary tube placed inside a rotating capillary tube

This was done in order to produce data on the kinetics of protofilament stabilisation immediately after the addition of GTP, which is not possible with existing techniques.

However, while doing the control experiments I found tha FtsZ protofilament formation was sensitive to the amount of dissolved air in the solution, and I also found that the FtsZ solution was sensitive to the mechanical agitation such as the Couette flow that occurs in LD cells. Neither of these characteristics appears to have been documented previously.

I found that degassing the solutions reduced this sensitivity both to the amount of gas present and also to mechanical agitation. This may help reduce the well known problems with obtaining consistent results with FtsZ.

The new LD cell was used to produce a small number of kinetics measurements of FtsZ protofilament formation. The results show that the new cell is able to produce LD spectral and kinetics data, but that there are problems with protocol robustness and measurement repeatability that would need to be investigated before this technique could be used to produce consistent and reliable LD measurements.

Introduction

During mitosis of prokaryotes a ring of proteins, the Z-ring, forms on the inside of the cell membrane where it will draw the cell membrane in, until it ultimately pinches off and the process of cell division is complete.

One of the first proteins to gather at the Z-ring is FtsZ, where it is subsequently joined by proteins such as ZipA, ZapA and FtsA which help stabilise the Z-ring, such that further proteins can build on a stable Z-ring and complete the process of mitosis (Margolin 2005). When the ZapA gene was originally identified as an unknown gene in Escherichia coli it was designated ygfE. The association of the homologue in B. subtilus with the Z-ring was subsequently determined (Gueiros-Filho and Losick 2002) and the protein named ZapA, and the gene zapA. This nomenclature has now been adopted increasingly for all the homologues of this gene and protein, including those in Escherichia coli.

In mini-project 1 approximately 100 mg of FtsZ were expressed and purified in order that various biophysical techniques could be used to investigate how these proteins stabilise the Z-ring.

It was intended that Linear Dichroism (LD) be used as one of these techniques. One of the drawbacks of the existing LD cell construction is that reaction components have to be mixed prior to the cell being assembled and the start of any measurements, which results in a lack of data for the first 10 seconds or so of the reaction when measuring reaction kinetics. A new cell design based on nested capillary tubes had been proposed to overcome this problem, and the first part of the mini-project was the additional development and characterisation of the cell to enable it to be used to investigate FtsZ stabilisation.

Prior to any investigations into the stabilisation of FtsZ by ZapA, some control experiments were conducted to verify the characteristics of the purified FtsZ and its use within the LD cell. Although it was possible to duplicate many of the experimental results previously obtained with FtsZ, it was found that FtsZ was sensitive to mechanical stress such as that induced by sonication or shearing within the LD cell, a characteristic previously unreported. This characteristic was believed to be responsible for the problems with the control experiment performed prior to investigating FtsZ using LD.

It was shown that LD and light scattering measurements are dependent on the amount of air dissolved in the polymerisation buffer used, and consequently the buffer solutions were degassed to reduce variation due to dissolved air. This also reduces the sensitivity to mechanical stress due to sonication.

Some LD kinetic and spectra measurements were obtained with the new cell, confirming that this cell may provide the basis for kinetic LD studies, including further studies on FtsZ. There are problems however with the reliability of the procedure, and the accuracy with which the solutions can be combined that require further study.

The time taken with validating the new measurement techniques, and the investigation into mechanical sensitivity meant that it was not possible to perform the intended biophysical investigations into the interactions between FtsZ and ZapA.

On this web site you will find:

• The methods and materials associated with this work
• Some experimental results
• More detailed discussion about the results
• Information on the design of the new LD cell.
• Initial test results using the cell.

In addition to the biophysical studies a review of existing structural data relating to FtsZ and its stabilising proteins was undertaken which I have written up. This includes investigations into the relationship between the electrostatic surface potentials of FtsZ and its stabilising proteins and the surrounding water and how this might relate to the interactions between these proteins and the experimental results obtained in this mini-project and previous studies.

Acknowledgements

I would like to thank my supervisor for this mini-project, Prof. Alison Rodger, together with my supervisor for my previous and linked mini-project, Dr. David Roper for their help and assistance.

I would also like to thank others who have provided facilities and their time to help with various parts of the project. These include Dr. Tim Dafforn from Birmingham University, who provided the use of the Analytical Ultra Centrifuge and kindly analysed the results, Dr Corinne Smith from Warwick Biological Sciences who provided access to the Transmission Electron Microscope, and Rhod Mortimore at Crystal Precision Optics for expertly grinding a quartz capillary from 3 to 2.7 mm diameter.

I would like to thank the other staff at Warwick who have provided help and advice, including Dr Matthew Hicks in Chemistry and Monica Lucena in the MOAC doctoral training centre.

I would also like to thank the many people in the Biological Sciences and Chemistry labs who have patiently taught me countless techniques, guided me through the processes and systems for making things happen, and showed me where things were kept, and how to get more when they run out. These include Martyn Rittman, Emma Gilroy (particularly for her suggestion to reduce the GTP concentration to overcome an accuracy problem with the new LD cell), Daniel Waldron and Min Zhang.

And I would particularly like to thank Raul Pacheco-Gomez, whose PhD marked the path for me to follow in many aspects of this mini-project, and often personally guided me along the path.

Finally, I would like to thank the Engineering and Physical Sciences Research Council (EPSRC) for providing the funding for the 4 year MOAC MSc/PhD that provides the context for this mini-project.