High Sensitivity Cryoprobe Equipment for the NMR Facility of the Department of Biochemistry University of Cambridge

NMR spectroscopy has emerged as a powerful tool to study the structure and mobility of biomacromolecules such as proteins and nucleic acids in their natural environment in solution. A particularly powerful feature of NMR is that interactions of molecules can be studied at an atomic level that allows to differentiate e.g. functional from structural regions in a protein and permit to characterize which residues are involved in a interaction at molecular level. Strengths and timescales of such interactions can be determined which reveal information about the underlying physical mechanisms behind such interactions. Unfortunately, NMR spectroscopy is a relatively insensitive method so that the majority of currently available studies have been limited to smaller proteins < 20 kDa. More recently novel techniques and improved spectrometer hardware have led to a dramatic increase in the basic sensitivity so that the study of larger biomacromolecules has become possible. One such significant advance has been the introduction of the so-called cryoprobe that improves the sensitivity of NMR experiments between two and five-fold. This dramatically higher sensitivity can be used to good advantage, to study larger proteins and their complexes with other proteins or DNA/RNA typically not accessible with conventional equipment, to investigate less abundant or aggregating proteins, or to reduce the time required to perform a particular NMR experiment, thus increasing the capacity of the spectrometer. Our Departmental NMR facility forms a collaborative research environment that serves a wide range of users from the Departments of Biochemistry and Chemistry that work on a multitude of structural projects. Many of these projects involve work with large proteins, or multi-protein complexes or have to be conducted at low protein concentrations so that this work would dramatically benefit from the availability of such cryoprobe equipment. In this application we are requesting funds to install cryoprobes on two out of our four facility machines to be able to pursue our proposed research projects. Some of our outlined projects are extremely demanding and high sensitivity NMR equipment is a pre-requisite for their feasibility. Here are some selected examples of biological and clinical importance from our research plan. Our areas of work cover structural studies of molecules and complexes involved in many aspects of cell-signalling and their implications in cell motility and cancer and the study of cancer susceptibility syndrome proteins involved e.g. in Fanconi Anaemia, a disease that leads to progressive bone marrow failure. Further projects study different aspects of misfolding of proteins such as can be found in plaque in brain tissue of patients that have the debilitating diseases of Alzheimer's or Parkinson's. The study of chromatin, central to the control of gene expression, it's interactions with DNA and many proteins is another major significant research area. Structural studies on intact ribosomes, the cellular centers where protein synthesis occurs, are relatively new and particularly challenging to perform due to the very low concentrations and limited lifetimes of the synthesized nascent protein attached to the ribosome. As a final example we mention the structural study of membrane proteins. These proteins account for ca. 30% of all encoded proteins and control a vast range of biological functions such as respiration, signalling and transport at a molecular level. As they are water insoluble structural information is hardly available. NMR can study such proteins in a detergent solubilized form but the large size of such complexes requires the highest NMR sensitivity available.

The aim of this application is to equip the NMR research facility of the Department of Biochemistry with a complete high sensitivity cryoprobe for the 800 MHz NMR spectrometer and to obtain a cryoplatform for an already purchased 500 MHz cryoprobe. Low sensitivity is one of the major limiting factors of NMR spectroscopy in the structural study of biomolecules. Modern cryoprobes increase the sensitivity of conventional equipment by a factor of two to five-fold (depending on salt concentration). The sensitivity of e.g. a 500 MHz system therefore becomes comparable to a 900 MHz albeit at a small fraction of its price (< 10%). Both spectrometer systems are part of the Departmental NMR facility that operates as a collaborative environment for the structural biology groups of the Departments of Biochemistry and Chemistry. Our proposed program of work contains a large amount of competitive projects that can only be adequately addressed in a high sensitivity NMR environment. The dramatically increased sensitivity will benefit the various structural studies of chromatin proteins, integral membrane proteins, proteins involved in DNA damage response and genomic instability syndromes, protein interactions involved in cell motility, invasion and metastasis, the study of protein folding and misfolding and disease related aggregation processes such as amyloid formation, modular synthetic enzyme systems and many more. The choice of an 800 MHz cryoprobe aims at achieving the best sensitivity on our highest resolution machine. This magnetic field strength provides for us the biggest TROSY improvements so crucial for high molecular weight work and further gives by far the best quality spectra from which we can obtain distance information and derive intermolecular structure data on complexes. All structural projects will benefit from the vastly enhanced performance. The 500 MHz cryoprobe will be used as a high sensitivity tool for triple-resonance experiments and mapping studies.