This grant proposes to develop the use of pseudocontact shifts in NMR spectroscopy for the determination of macromolecular structure. Pseudocontact shifts, which result from the presence of paramagnetic metal ions, will be used as constraints in a molecular simulation program, together with empirical force fields, to define the coordinates of the resonating nuclei. An initial test on cytochrome c demonstrated excellent agreement between observed and calculated shifts, and extremely high precision of coordinate determination. The use of positional rafter than distance based constraints allows for the incorporation of experimentally determined motional parameters such as order parameters in NMR. A picture of conformational flexibility is essential to an understanding of protein function, and is not directly included in current NMR or crystallographic analysis. The methods will be developed and demonstrated on several systems for which structural features of interest are not defined sufficiently by the NOE. 1) DNA oligomer complexed with cobalt and chromomycin will be investigated. The use of long range pseudocontact shifts is particularly attractive in such a system, where only a limited number of short interproton distances occur. 2) Lanthanide induced shifts in a calcium- binding protein, calbindin, will be analyzed, with athe aim of defining more precisely several loops of the protein, and demonstrating the use of heteronuclear pseudocontact shifts. The use of lanthanide induced shifts for quantitative structure determination looks promising given rapid advances in the fields of both computational chemistry and NMR spectroscopy. 3) Pseudocontact shifts in zinc fingers with cobalt substitution for zinc will be studies for their potential in athe definition of zinc fingers and their complexes with DNA or RNA. A clear understanding of molecular structure is important for unraveling the complex interactions of biologically important molecules. Metal ions are intricately involved in many systems, including interactions between proteins and drugs with nucleic acids, maintenance of the structural fold in metalloproteins and ribozymes, and determination of function in metalloenzymes. The use of these will situate metals as spectroscopic probes will enhance our understanding of macromolecular structure.
