With the availability of complete genomes, protein structure elucidation becomes increasingly important as a tool for predicting protein function. NMR provides a powerful solution technique for structure determination. However, conventional techniques are often labor and time intensive. New approaches, which minimize the time required to calculate a protein global fold are necessary to increase the effectiveness of NMR in global fold identification. This proposal will develop paramagnetic techniques for rapid protein global fold elucidation. Paramagnetic techniques can provide distance restraints and angular restraints. The use of site-directed spin and isotope labeling can provide distances up to 35A between the covalently attached spin label and backbone protons. Angular restraints obtained from partial alignment of the target protein in the magnetic field can provide a wealth of information on both the secondary and tertiary structure of the protein. Residual dipolar couplings, dipolar shifts, and information obtained from cross-correlation between Curie spin and dipolar relaxation provide restraints for global fold identification. To date, the general utility of these techniques in protein global fold recognition is largely under-appreciated. Finally, a rapid method for introduction of metal binding tags into target proteins will be developed. Initially, the zinc finger domain of the retroviral gag protein will be used. This peptide binds both zinc and cobalt with high affinity. The zinc finger domain is fused to either the or C- terminus of the target protein. Incorporation of cobalt into the metal-tag fusion protein induces partial alignment of the protein in the magnetic field. The proposed methodology offers a number of advantages. Rapid affinity purification protocols can be developed based on the metal binding tag. Finally, introduction of metal binding tags at both the and C- termini of the protein should introduce different orientations of the protein in the magnetic field.
| Dvoretsky, Alex; Gaponenko, Vadim; Rosevear, Paul R (2002) Derivation of structural restraints using a thiol-reactive chelator. FEBS Lett 528:189-92 |
