Copper-containing metalloenzymes constitute a major class of proteins that catalyze a myriad of reactions in nature. Inspired by the structural and functional characteristics of this unique class of metalloenzymes, we report the conception, design, characterization, and functional studies of a de novo artificial copper peptide (ArCuP) within a trimeric self-assembled polypeptide scaffold that activates and reduces peroxide. Using a first-principles approach, the ArCuP was designed to coordinate one Cu via three His residues introduced at an a site of the peptide scaffold. X-ray crystallography, UV–vis, and electron paramagnetic resonance data demonstrate that Cu binds via the Nε atoms of His forming a T2Cu environment. When reacted with hydrogen peroxide, the putative copper-hydroperoxo species is formed where a reductive priming step accelerates the rate of its formation and reduction. Mass spectrometry was used to identify specific residues undergoing oxidative modification, which showed His oxidation only in the reduced state. The redox behavior of the ArCuP was elucidated by protein film voltammetry. Detailed characterization of the electrocatalytic behavior of the ArCuP led us to determine the catalytic parameters (K M and k cat), which established the peroxidase activity of the ArCuP. Combined spectroscopic and electrochemical data showed a pH dependence on the reactivity, which was optimum at pH 7.5.
E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters of ensconsin distribution and dynamics; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells.
We report the construction of an artificial hydrogenase (ArH) by reengineering a Cu storage protein (Cspl) into a Ni-binding protein (NBP) employing rational metalloprotein design. The hypothesis driven design approach involved deleting existing Cu sites of Csp1 and identification of a target tetrathiolate Ni binding site within the protein scaffold followed by repacking the hydrophobic core. Guided by modeling, the NBP was expressed and purified in high purity. NBP is a well-folded and stable construct displaying native-like unfolding behavior. Spectroscopic and computational studies indicated that the NBP bound nickel in a distorted square planar geometry that validated the design. Ni(II)-NBP is active for photo-induced H 2 evolution following a reductive quenching mechanism. Ni(II)-NBP catalyzed H + reduction to H 2 gas electrochemically as well. Analysis of the catalytic voltammograms established a proton-coupled electron transfer (PCET) mechanism. Electrolysis studies confirmed H 2 evolution with quantitative Faradaic yields. Our studies demonstrate an important scope of rational metalloprotein design that allows imparting functions into protein scaffolds that have natively not evolved to possess the same function of the target metalloprotein constructs.
Transition-metal-catalyzed oxidative stress is a widespread concern in the pathogenesis of Alzheimer's disease. However, the exact role of amyloid beta oligomers towards oxidative stress is widely debated. Assessing the oxidative nature of the oligomers in vitro is complicated by the different experimental conditions under which they are prepared. We have investigated Cu -catalyzed reactive oxygen species (ROS) generation by using oligomers prepared in phosphate-buffered saline (Aβ ) and in cell culture medium (Aβ ), and compared their activities with respect to the monomers and fibrils prepared at neutral and acidic pH. Although both are deca- to dodecamers, the Aβ oligomers have a spherical morphology and are smaller than the Aβ . The Aβ behaved as pro-oxidants; in contrast, Aβ quench OH generation attributed to CCM itself. Although the pro-oxidant oligomers showed oxidation, they also partially protect themselves from radical damage and maintain their overall spherical arrangement. The monomers and fibrils manifested antioxidant properties: radical scavenging as opposed to redox silencing. A dual role of Aβ species depending on the stage of the disease is proposed. In the earlier stages, the monomers can act as antioxidants, whereas at the later stages, the oligomers take on a pro-oxidant role. Kaempferol, a natural flavonoid, bound Cu in 2:1 ratio and abolished ROS production in all Aβ species. It also distinctly modified the folding landscape of Aβ species into new or altered morphologies.
Hydrogeni saclean and sustainable form of fuel that can minimize our heavy dependence on fossil fuels as the primary energy source.T he need of finding greener ways to generate H 2 gas has ignited interest in the research communityt os ynthesize catalysts that can produce H 2 by the reduction of H + .T he natural H 2 producing enzymes hydrogenases have served as an inspiration to produce catalytic metal centers akin to these native enzymes. In this article we describe recent advances in the design of au nique class of artificial hydrogen evolving catalysts that combine the features of the active site metal(s) surrounded by ap olypeptide component. The examples of these biosynthetic catalysts discussed here include i) assemblies of synthetic cofactors with native proteins;i i) peptide-appended synthetic complexes;i ii)substitution of native cofactors with nonnative cofactors;i v) metals ubstitution from rubredoxin;a nd v) ar eengineeredC us torage protein into aN ib inding protein. Aspects of key design considerations in the construction of these artificial biocatalystsa nd insights gainedi nto their chemical reactivity are discussed.
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