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dc.contributor.advisorJackson, Timothy A.
dc.contributor.authorGlass, Amanda M.
dc.date.accessioned2012-11-26T21:38:01Z
dc.date.available2012-11-26T21:38:01Z
dc.date.issued2012-08-31
dc.date.submitted2012
dc.identifier.otherhttp://dissertations.umi.com/ku:12273
dc.identifier.urihttp://hdl.handle.net/1808/10440
dc.description.abstractThe tripeptide asparagine-cysteine-cysteine (NCC) was found by the J. Laurence lab to bind nickel very tightly. In fact, NCC was serendipitously discovered as a metal-abstraction peptide tag (MAP-tag) because it was stripping nickel from a standard immobilized metal affinity chromatography (IMAC) resin while the Laurence lab was making a protein previously unknown to bind metals. Given the ability of NCC to bind nickel, the MAP tag has the potential to be a useful biotechnological metal-delivery agent. However, prior to this work, the structure and chemical reactivity of the nickel-peptide complex were undefined. The work presented herein involves structural and reactivity investigations of the MAP-tag bound with NiII in the dianionic complex [Ni-NCC]2-. A series of spectroscopic tools, including electronic absorption, circular dichroism (CD), magnetic CD (MCD), variable-temperature variable field (VTVH) MCD, and X-ray absorption (XAS), as well as density functional theory (DFT) and time-dependent (TD) DFT methods were used to provide insight into the structure of the complex. A summary of the significant findings in this work is provided below. Spectroscopic and computational data collected of Ni-NCC conclude that NiII ion is bound in a 2N:2S square plane with coordination by the N-terminal amine nitrogen, the deprotonated backbone amide nitrogen of Cys2, and two cysteinate sulfur atoms from Cys2 and Cys3. This ligand environment is very similar to that of the nickel ion bound in the protein nickel superoxide dismutase (Ni-SOD), and comparison of the electronic absorption and CD spectra confirms that Ni-NCC and Ni-SOD have the same nickel primary coordination sphere. In addition, Ni-NCC is able to consume superoxide. Therefore, Ni-NCC is both a structural and functional mimic of Ni-SOD and detailed analysis of the electronic and geometric structures of Ni-NCC can provide insight into the minimal unit necessary to afford activity in the enzyme. Over time, the complex LLL-NiII-NCC undergoes site-specific chiral inversion to convert to DLD-NiII-NCC, as determined by spectroscopic analysis of authentic D-containing Ni-NCC peptide complexes and supported by DFT single point energy computations on geometry optimized models of the complex. This structural rearrangement enhances the superoxide scavenging ability of Ni-NCC. Intriguingly, this chiral inversion is not observed in either Ni-SOD or any small molecule mimics of the enzyme, and is therefore unique in this mimic. Upon evaluating the DFT-computed models, this rearrangement of LLL-NiII-NCC to DLD-NiII-NCC exposes one face of the nickel ion in the complex. This may allow for better interaction with exogenous molecules, like CN- and O2.-. Thus, rearrangement within the complex increases superoxide consumption. Although other metal-peptide complexes are known to undergo metal-facilitated, base-catalyzed chiral inversion or racemization, the chiral inversion reaction of Ni-NCC does not demonstrate first-order dependence on hydroxide ion concentration. Therefore, the pathway to chiral inversion in Ni-NCC is distinct from other metal-peptide complexes. Although the chiral inversion reaction of LLL-NiII-NCC to DLD-NiII-NCC is minimally affected by solution pH, the reaction is absolutely dependent on the presence of O2. We propose that O2 is required to oxidize the metal center to generate NiIII-NCC, which is then able to undergo the inversion reaction. Generation of superoxide, as well as NiIII, during the aging of LLL-NiII-NCC supports this hypothesis. Thiolate coordination in NiII-NCC reduces the effective nuclear charge of the nickel ion, necessitating oxidation to LLL-NiIII-NCC before the chiral inversion reaction can be initiated. Additionally, DLD-NiII-NCC also undergoes a small extent of O2-dependent reactivity. However, spectroscopic characterization confirms that DLD-NiII-NCC reaches the same final state as LLL-NiII-NCC. In order to characterize the structure of authentic, O2-free, LLL-NiII-NCC and DLD-NiII-NCC, as well as the O2-exposed forms of the peptides, Ni K-edge XAS was employed. This characterization of authentic, O2-free LLL-NiII-NCC and DLD-NiII-NCC confirmed the proposed 2N:2S square planar coordination in NiII-NCC. Similarities of the pre-edge feature(s), edge energy, and radial distances determined for NiII-NCC with NiII-SOD and other NiII-SOD peptide mimics further supports that Ni-NCC is a structural mimic of the enzyme. In addition, XAS characterization of O2-exposed LLL-NiII-NCC and DLD-NiII-NCC shows that, even during the chiral inversion reaction of Ni-NCC, the immediate structure about the nickel center does not significantly change.
dc.format.extent156 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
dc.subjectInorganic chemistry
dc.subjectBiochemistry
dc.titleCHARACTERIZATION OF THE STRUCTURE AND REACTIVITY OF A NICKEL(II)-TRIPEPTIDE MIMIC OF NICKEL SUPEROXIDE DISMUTASE
dc.typeDissertation
dc.contributor.cmtememberBarybin, Mikhail V.
dc.contributor.cmtememberBenson, David R.
dc.contributor.cmtememberLaurence, Jennifer S.
dc.contributor.cmtememberScott, Emily E.
dc.thesis.degreeDisciplineChemistry
dc.thesis.degreeLevelPh.D.
kusw.oastatusna
kusw.oapolicyThis item does not meet KU Open Access policy criteria.
kusw.bibid8085830
dc.rights.accessrightsopenAccess


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