100 kHz magic angle spinning for development of solid-state NMR methodology for probing protein dynamics

Motion and change are essential features of living organisms and fundamentally important for many vital processes from protein folding and unfolding, ligand binding, signalling, allosteric regulation to enzymatic catalysis. Consequently, understanding motions at molecular level provides valuable insights into the phenomena involving change of structure both when they function as intended or when they malfunction. For example understanding how proteins misfold may help to fight debilitating diseases called amyloidoses that include Alzheimer's disease, type II diabetes or bovine spongiform encephalopathy more widely known as "mad cow" disease. Moreover, understanding motions that are intrinsically associated with signalling pathways may result in development of better drugs that target such pathways (most medicines work this way). Even development of practical environmentally friendly biobatteries and biofuel cells may be aided by knowledge of molecular motions as they make use of enzymes. Thus it is really important to devise ways to measure protein motions at atomic resolution.
To do that, in this project, we will develop a technique called nuclear magnetic resonance (NMR), which relies on the inherent magnetism of atomic nuclei. When placed in a strong magnetic field magnetic moments of nuclei align with the external field but this alignment may be changed by application of radio waves at specific frequencies. By measuring the associated frequencies one can learn about the relative position of atoms with respect to each other and how this position changes with time i.e. molecular motions. A very powerful aspect of this technique is that one can learn such information not only for a molecule overall but for specific atoms in it. In solid-state NMR, which is the primary method used in this project, the high resolution necessary to distinguish individual sites is enabled by a technique called magic angle spinning (MAS), which involves fast rotation of the sample around an axis inclined at an angle of 54.7 degrees to the external magnetic field. Recently introduced cutting edge instrumentation allows achieving spinning frequencies up to 100 000 revolutions per second. The centre of this project is the purchase of the first in the UK probe capable of 100 kHz MAS. The improved efficiency of MAS at such astounding frequencies makes possible designing new experiments that provide new analytical tools to access motions, e.g. site-specific 1H relaxation or highly sensitive 1H-detected relaxation measurements in fully protonated samples. The 100 kHz spinning removes a number of undesired effects obscuring the measurements of parameters reporting on molecular motions and thus allows a detailed view of protein motions to be obtained.
In this project we propose to develop a series of robust solid-state NMR spectroscopic methods that take advantage of the new 100 kHz spinning regime and will provide improved access to measuring of dynamic processes in proteins at atomic resolution and in a site-specific manner. In particular, we will focus on techniques that provide access to slow motions in the regime that is difficult to access by the solid-state NMR sister method - solution NMR. In addition, in order to improve practicality of the developed techniques we will optimise them for speed and sensitivity.

Impact through training
Three PhD students will be trained in developing NMR methodology at 100 kHz MAS as a part of this research project. The transferable skills and unique analytical skills obtained as a result of participating in this research will facilitate progress of their main PhD projects and will improve their employability in biotech industry or academia.

Impact through collaborations with industry
The PI has established two industrial collaborations that will directly benefit from this project. The collaboration with Bruker involves developing methodology that helps to preserve viability of biological samples under the conditions of extremely fast magic angle spinning which is directly relevant to this project. In addition, the main project of one of the PhD students involves industrial collaboration with Pfizer on application of NMR relaxation based methods to facilitate chromatographic method development and probing stability of pharmaceutical formulations. This collaboration will be used as a platform to transfer the methodology developed in the context of fundamental biomolecular studies to practical industrial applications including optimisation of compound separation, characterisation of pharmaceutical formulations and drug development.

Impact on technology development
As this project involves efforts to maximise the utility of novel fast magic angle spinning technology it will lead to popularisation of such technology and provide motivation for further technological development. Currently, there are competing manufacturers developing such technology.

Societal and environmental impact through scientific progress
The tools developed as a result of this project will allow characterising in detail dynamic transformations of proteins that are implicated in their function. Consequently, such tools may aid understanding of many diseases involving dynamic changes of proteins and in a longer-term help finding a cure for them and thus contribute to enhancing quality of health and life of the society. For example, knowledge of dynamics of protein kinases should help in developing their effective inhibitors and thus aid treating certain forms of cancer. In general, dynamical factors need increasingly to be considered in addition to structural factors in order to design effective ligands and inhibitors of proteins and hence effective drugs. The tools developed in this project will facilitate this process. These tools can also contribute to understanding processes of protein misfolding that are at the core of diseases such as Alzheimer's or type II diabetes that are becoming an increasing burden on the UK and other health systems around the globe. Finding a cure for these diseases facilitated by research enabled by our results can have profound health and economic impact on the society.
Mobility is important for the function and stability of enzymes. Consequently, any processes involving use of enzymes can benefit from tools enabling detailed characterisation of protein motions: from industrially important immobilised enzymes to developing and perfecting environmentally friendly biobatteries and biofuel cells and facilitating rational synthetic biology approaches to produce new drugs difficult to synthesise by traditional chemical means.
In order to ensure that our results reach the right scientific audience we will use the traditional dissemination methods such as publications in international peer-reviewed journals and presentations at conferences as well as direct transfer of knowledge through collaborations with researchers working on the discussed above issues (see Case for Support and Pathways to Impact).