Designing protein separations based on metal-affinity interactions

• Main Author: Todd, Robert J.
• Format: Others
• Language: en
• Published: 1993
• Online Access: https://thesis.library.caltech.edu/4690/1/Todd_rj_1993.pdf; Todd, Robert J. (1993) Designing protein separations based on metal-affinity interactions. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/aph0-0p32. https://resolver.caltech.edu/CaltechETD:etd-11292007-090410 <https://resolver.caltech.edu/CaltechETD:etd-11292007-090410>

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. Immobilized metal-affinity chromatography (IMAC) provides a technique to separate proteins based on their histidine multiplicity and microenvironment. A set of S. cerevisiae iso-l-cytochrome c variants which differed only in their histidine content and placement was used to study equilibrium binding and chromatographic retention on matrices of immobilized Cu(II) (TSK macroporous support). The structural integrity of the variants was verified by UV-visible absorbance and [...] NMR spectroscopy. Equilibrium binding studies indicate that a surface histidine is required in iso-l-cytochrome for interaction with a Cu(II)IDA (iminodiacetic acid) matrix. The apparent binding affinity of variants depends on the accessibility of the histidine side chains and on the total number of surface histidines. The overall equilibrium binding behavior is consistent with a model in which bound protein covers multiple copper sites on the matrix surface. Chromatographic studies on a Cu(II)IDA TSK column showed that a surface histidine is required for significant protein retention. It was also shown that proteins can be separated based on the microenvironment (accessibility or [...]) of a single surface accessible histidine or on the multiplicity of surface histidines and the order of retention in all cases correlated with the initial slope of the equilibrium binding isotherm. The effect of proton and imidazole competition on retention was also studied, and it was seen that simple equilibrium models, describing the interaction of a single-histidine protein with independent copper binding sites, are insufficient to account for the strong competitive behavior that is observed experimentally. The ability of IMAC to discriminate between proteins based on their histidine multiplicity and microenvironment provides a powerful dimension in protein separations. The concept of engineering di-histidine metal-chelating sites into proteins for applications in purification and protein stabilization was investigated. A His-[...]-His site was engineered into the N-terminal [...]-helix of iso-l-cytochrome c at positions 4 and 8. A di-histidine site was also engineered across a small section of [...]-sheet structure at positions 39 and 58. Cu(II)IDA titrations of [...] NMR spectra show selective paramagnetic broadening of the C2 resonances of the chelating histidines. The His-[...]-His site at positions 4 an 8 binds Cu(II)IDA-PEG in solution 24 times stronger than a single histidine site and both metal-chelating variants have increased retention in a Cu(II) IMAC column relative to a non-chelating control protein. The engineered metal-chelating sites were also shown to stabilize the folded form of iso-1-cytochrome c in the presence of Cu(II)IDA by as much as 2.5 kcal/mol.