Measuring Nanometer Scale Distances by high-field Pulse Electron Paramagnetic Resonance Using Mn(II) Spin-Labels

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Techniques for measuring nanometer-scale distances have become invaluable tools in biology, how large conformational dynamics and macromolecular interactions determine cellular process has become a new paradigm. Pulse electron double resonance (PELDOR) spectroscopy has proven to be extremely useful in this regard. The subject of this proposal is the development and application of high microwave-frequency (35 to 180 GHz) high-magnetic fields (up to 6 Tesla) PELDOR using Mn(II) ions as spin-labels. High-field PELDOR has principally two advantages: (1.) much smaller samples volumes (2 μL) and (2.) the potential for achieving higher resolution angle measurements. To realize these benefits, new spectroscopic and chemical approaches will be required. Stable nitroxide radicals are the spin-labels of choice for conventional 9 GHz (0.3 T) PELDOR measurements, but they are far from optimal for use at higher magnetic-fields. By contrast, Mn(II) ions have a magnetic-field dependence that makes them far more attractive as high magnetic-field PELDOR spin-labels. Although the EPR spectroscopy of spin-5/2 Mn(II) ions is as well-defined as S=1/2 radicals, it is more complex than that of radicals. Therefore, a major goal of this project is to understand how this affects PELDOR measurements and to develop approaches to address and even exploit this complexity. To achieve greater utility, the second major goal of this project is to develop Mn(II) spin-label technology so that Mn(II) PELDOR can be generalized. Initially, robust Mn(II) bis-terpyridine complexes will be attached to model systems and biological complexes using chemistry similar to that used for radicals. Other complexes and linking chemistries will be explored as the technique is progressively refined. The Mn(II) PELDOR will be tested on and used to study both model and biological systems that are “naturally labeled”, that is metalloenzymes that naturally bind Mn(II) ions, and those labeled synthetically using technology that will be developed.