High field ENDOR of metal centers in proteins: Applications and methodology

• Applicant: Professorin Dr. Daniella Goldfarb
• Subject Area: Analytical Chemistry
• Term: from 2002 to 2004
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5386263

Transition metal ions are essential components in many systems with special functions and the structural properties of the metal sites are usually closely related to their specific activity. The details of this relation are, however, not fully comprehended. Therefore, a complete characterization of such sites in terms of the spatial organization of the atoms and the electronic structure is important. Such information can be obtained from EPR spectroscopy based methods provided that the metal ions are paramagnetic. The recent developments of high field EPR in general, and high field electron-nuclear double resonance (ENDOR) spectroscopy in particular, offer unique opportunities to study paramagnetic metal active sites in both orientationally disordered systems and in single crystals. The latter are usually too small for conventional X-band EPR/ENDOR studies. In the last four years of the project we have conducted a detailed W-band ENDOR study of the Mn(II)(S=5/2) space binding site of the metalloprotein concanavalin A (conA) in frozen solutions and in single crystals, and have detected in the crystal the appearance of two chemically inequivalent Mn(II) upon cooling. This escaped detection by low temperature high resolution X-ray diffraction. The objectives for the next two years are: (i) Use the knowledge we acquired from the conA study we carried out earlier and apply it to Mn(II) active sites in the enzyme enolase. (ii) Develop and apply two-dimensional (2D) methods that will facilitate assignment of ENDOR peaks, will enhance resolution and will provide new correlations and constraints that can be translated into structural information. (iii) Investigate experimentally and theoretically the characteristics of ENDOR effect and their implications on the observed lineshapes at low temperatures and high fields (large thermal polarizations).

• DFG Programme: Priority Programmes
• Subproject of: SPP 1051: Hochfeld-EPR in Biologie, Chemie und Physik
• International Connection: Israel