Hydrogen-1 nuclear magnetic resonance investigations of the paramagnetic derivatives of the metalloproteins parvalbumin, fixl, and aminopeptidase

Nuclear Magnetic Resonance (NMR) at 360 MHz has been successfully used for the study of metalloproteins ranging in molecular weights from 12 to 31 kDa and containing a variety of metal binding sites. Typically useful information cannot be obtained on biomolecules of this size using magnetic fields lower than 500 MHz. However, this work was made possible by the use paramagnetic metal ion substitution to shift signals of protons in the metal binding sites outside the diamagnetic region of the 1H NMR spectrum. Though the line broadening caused by the presence of unpaired electrons renders traditional 1H NMR analysis of proteins impossible, we have implemented signal assignment strategies to identify amino acid residues and ligands directly coordinated or close to the metal binding sites.; Singly and doubly Yb3+-substituted derivatives of parvalbumin isolated from northern pike have been successfully prepared and characterized by the use of optical and 1H NMR spectroscopies. By the use of paramagnetic lanthanide ion substitution of the native Ca2+ ion, signals of amino acid residues in close proximity to both metal binding sites were detected and assigned by use of 2D NMR techniques such as COSY, NOESY, and TOCSY.; The cyano-met form of the truncated heme region of the oxygen sensor FixL from Rhizobium meliloti has been characterized by the use of 1D and 2D 1H NMR techniques. Through ligation of CN −, the spin state of the native Fe3+ was changed from high-spin to low-spin, allowing the signal assignment of the heme ligand and proximal histidine residue. The unique spectral features and the lack of signals for a distal histidine suggest that this heme protein is quite different from all other heme proteins such as the globins and cytochromes. This work represents the first 1H NMR data obtained on a new class of heme proteins known as the oxygen sensors.; Cu2+ and Co2+-substituted derivatives of aminopeptidase from Streptomyces griseus were prepared to determine the mode of Ca2+ activation and phosphate inhibition of this enzyme. The di-Cu2+ derivative shows strong magnetic coupling allowing signal assignment. Upon addition of Ca2+ ion, it is seen that the signals of Tyr246 and Asp160 are broadened suggesting a decrease in the metal-proton distance of these residues. Co2+ has been shown to bind sAP selectively, allowing the preparation of a singly substituted derivative CoE-sAP. 31P-NMR relaxation was then measured on this sample as a function of phosphate concentration to determine the distance between the paramagnetic metal and the phosphate. A calculated distance of ≥4.03 Å suggests that phosphate does not coordinate directly to the metal site as was reported in the crystal structure.