Collection and interpretation of NMR structural information is often time-consuming, while the demand of pharmaceutical industries for structures of newly discovered proteins is high. This research demonstrates the utility of paramagnetic probes for the rapid determination of protein global folds using paramagnetic relaxation enhancements, dipolar shifts, and dipolar couplings.
Attachment of nitroxide and thiol-reactive EDTA tags to unique cysteines allowed use of the relaxation effects of unpaired electrons in nitroxides and manganese ions on nuclei within the protein of interest for measurement of electron-nucleus distances. Distances obtained in nitroxide labeled mutants of barnase, H102C and Q15C, combined with secondary structure restraints permitted calculation of a barnase global fold with the 3Å root mean square deviation of backbone coordinates from the crystal structure of barnase.
Introduction of EDTA tags into the same barnase mutant proteins allowed the use of manganese-amide proton distances and cobalt induced dipolar shifts for calculation of amide proton atomic coordinates onto the z-axis and the xy-plane of the paramagnetic susceptibility tensor. In addition, the anisotropic magnetic field of cobalt induced alignment of protein molecules in the external magnetic field permitting measurement of dipolar couplings.
Manganese and cobalt were also incorporated into barnase by fusing a zinc finger tag to the protein C-terminus allowing determination of z-coordinates and measurement of dipolar couplings. Despite the significant amount of error introduced by the mobility of the tag, an accuracy of +/- 4.5Å in calculation of z-coordinates could be achieved.
The utility of unique paramagnetic reference frames for structure determination was demonstrated on calbindin containing calcium binding sites that could be substituted with cerium, ytterbium, and disprosium. Minimization against lanthanide induced dipolar shifts produced a protein structure with 3.3Å resolution. However, only 29 out of 76 amide protons in calbindin were used for global fold determination due to extreme line-broadening.
In conclusion, this research has demonstrated the utility of paramagnetic systems for rapid protein structure elucidation. It has been shown that paramagnetic probes can be incorporated into nonmetal-binding proteins. The use of structural restraints obtained using paramagnetic probes should permit rapid determination of global folds of newly discovered proteins.
|School:||University of Cincinnati|
|Department:||Medicine : Molecular Genetics, Biochemistry and Microbiology|
|School Location:||United States -- Ohio|
|Source:||DAI-B 79/10(E), Dissertation Abstracts International|
|Keywords:||Global fold, Labeling, Nmr, Paramagnetic systems, Protein structure|
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