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Incorporation of histidine-rich metal-binding sites onto small protein scaffolds: Toward developing direct Tc-labeling of proteins

¹ Dept. of Chemistry; ² Dept. of Radiology, University of Iowa, Iowa City, Iowa

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Objectives: Mutants of small, well-folded protein and peptide scaffolds were designed with preorganized, histidine-rich binding pockets resulting in local environments in the protein structure suitable for binding Tc(CO)₃⁺ without needing non-natural chelators and spacers.

Methods: Two mutants of ubiquitin (3HIU and 3HPU) were designed to incorporate a 3-histidine insertion with glycine spacers at residue 9 of the protein or three histidine point mutations near the 35-38 turn, respectively. In the "Trp-cage" mutant, two histidine point mutations were made on the N-terminal helix with the third histidine added in a 4-glycine N-terminal tail. [^99mTc(CO)₃(H₂O)₃]⁺ was made by standard procedures, then added to mutant proteins at a final protein concentration of 50 µM. ^99mTc-binding was verified by RP-HPLC. To test for protein/metal stability, free L-histidine was added to these ^99mTc + mutant reactions to a final concentration of 57 µM. Tc-protein complex stability was examined by RP-HPLC for Tc-99m release.

Results: All three mutants show binding of the [Tc(CO)₃]⁺ species. HPLC analysis showed that the [Tc(CO)₃]⁺ in the 3HIU and 3HTCb complexes is not labile in the presence of excess histidine. However, the 3HPU-Tc-99m complex was not stable to excess histidine.

Conclusions: Preliminary results show that proteins and peptides can be engineered to bind directly to Tc(CO)₃⁺ without the need for non-natural chelators. These experiments demonstrate the possibility of designing proteins and peptides with Tc-coordinating ligands incorporated into the protein structure.

Research Support: NSF, MCB-045119

This Article Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Soebbing, S. Articles by Franklin, S. PubMed Articles by Soebbing, S. Articles by Franklin, S.