Dynamic post-translational histone modifications studied by NMR spectroscopy

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DNA molecules of human cells are many times longer than the diameter of the cell and consequently the DNA is packed into a compact structure called the chromatin. The chromatin consists of DNA molecules coiled around histone proteins (sticky pulleys) in a very systematic manner. The cell utilises several mechanisms to control exactly what inheritable information from the DNA molecule that is turned into functional product (cellular machines). One mechanism that the cell exploits is to change the charge of certain histone proteins (weaken or strengthen the stickiness of the pulleys) and thus expose or restrict a specific part of the DNA to the cells gene production apparatus. HDAC, an enzyme that is responsible for changing the charge of histone proteins (a stickiness enhancer) will be the focus of the proposed research project. The HDAC enzyme works as a scissors that strips a negative charge off the histone proteins, thereby rendering the histone tails positively charged which strengthen the interaction with the negatively charged DNA. In particular, the focus of the proposed project is the dynamics and molecular motions of the HDAC enzyme (how does the scissors cut) and the dynamics will be studied primarily with nuclear magnetic resonance (NMR) spectroscopy. Thus, one of the key objectives of the research is to characterise, at atomic resolution, the mechanism by which the HDAC enzyme alter the histone charges. The goal is also to characterise how HDAC enzymes interact with inhibitors (drugs) and histones. HDAC enzymes are involved in cancers where they are believed to suppress the production of tumour suppressors. Inhibitors of HDAC enzymes have shown anti-tumour activity and it is therefore likely that the outcome of the proposed research will lead to the design of specific inhibitors of HDAC enzymes ultimately resulting in more efficient cancer therapy. An understanding of histone modifications requires a detailed picture of the three-dimensional structure of the involved enzymes and an appreciation of how these structures vary and fluctuate with time (a scissors cuts due to its opening and closing motions). Static structures of HDAC enzymes have been determined over the last decade, however, very few studies on the flexibility and dynamics of these regulatory molecules have been published. The proposed project focuses on the use of NMR spectroscopy as the primary biophysical tool to elucidate molecular flexibility and interactions since NMR has the potential to provide a description of the dynamics and interactions at atomic resolution. It is the goal that the NMR measurements together with other experimental techniques and computer simulations will create a coherent characterisation of the enzyme function. Another major objective of the proposed research is to develop new NMR methods to characterise molecular dynamics and flexibility in general. These developments aim at a time-resolved description of enzyme motions, that is, a visualisation of the enzyme motions over time - as a movie - as opposed to previous methods that primarily provides the amplitudes of protein motions. The research will be carried out at the Institute of Structural and Molecular Biology (ISMB), a joint venture between Birkbeck and University College London (UCL). UCL and ISMB provide a state-of-the-art and stimulating research environment with dedicated NMR machines suitable for the proposed project. Also, the highly collaborative environment and world-class expertise at ISMB and UCL open up the possibility for fruitful collaborations. For example, to increase the likelihood of success in the development of new HDAC inhibitors, I have initiated a collaboration with Prof Charles Marson, UCL, who is an expert on the productions of HDAC inhibitors. In my opinion this collaboration will allow the results about the HDAC enzyme dynamics, obtained by NMR spectroscopy, to be taken one important step further towards the design of new medicine.