Geometry Information from Paramagnetic NMR Relaxation

The main goal of this research is to introduce new and improve existing scientific tools and strategies for the investigation of the molecular structure of biologically active substances containing so-called radicals (or paramagnetic centers). For this purpose we will mainly make use of nuclear magnetic resonance (NMR) spectrometers. Paramagnetic substances (often referred to as "free radicals") play crucial roles in the processes of life, which may be either of advantageous or detrimental for the affected organism. It is therefore important to develop and test new methodology that gives better insight into the structure and behavior of this type of biomolecules. In this research project we intend to develop an refine methods and strategies that allow to determine the orientation of selected parts of a paramagnetic biomolecule relative to the position of the free radical is located. We will use methods based on NMR-technology, which has unique capabilities for structure studies in solution, but encounters some special challenges in the case of paramagnetic molecules. Refining the quantification of a physical effect (PIN, paramagnetic induced narrowing), which we recently discovered, and combining it with other recent sources of information on molecular structure from NMR, we want to extend the arsenal of tools and strategies available to investigate the three dimensional structure of paramagnetic molecules and their interactions in detail.

During this project we have developed new methodology to investigate the arrangement of atoms in a particular class of proteins, namely paramagnetic proteins, in solution by nuclear magnetic resonance (NMR) spectroscopy. Paramagnetic proteins are important in many processes in living cells that involve the transfer of energy such as respiration and photosynthesis. For this purpose a novel technology - a so-called cryogenically cooled probe - was for the first time installed and applied in Austria. This probe allows highly sensitive detection of NMR signals - ca. four times more sensitive than the best technology available previously. As a consequence many measurements require only 1/16 of the time previously necessary. Alternatively only a quarter of the sample amount is needed to obtain results in the same amount of time. This is essential for the application of NMR-techniques to dilute paramagnetic proteins. Based on this technology special measurement techniques and protocols were developed that are tailored to determine geometrical parameters in paramagnetic proteins. The technique has also been applied successfully to the determination of the structure of components of the bacterial cell wall. Here the high sensitivity of the cryo-probe technology allowed for the first time structure determination of minute amounts of polymeric carbohydrates by nuclear magnetic resonance without using isotopic enrichment techniques. Apart from the primary goal of molecular structure determination also some important unexpected results were obtained through the cryo-NMR technology. In particular the groundwork was laid for an entirely new image- generating technique, which allows, for example, to map the spatial distribution of water in a specimen placed in a weak magnetic field gradient, by just recording the radio frequency noise. This is a substantial advantage over previous techniques which require the use of intense electromagnetic radiation pulses to excite the signals, which have raised safety concerns.