De Novo Design of Copper Metallopeptides Capable of Electron Transfer: From Design to Function.

Biophysical characterization on de novo designed three-helical bundles is presented. The structure was determined to establish its physical integrity. Spectroscopic, electrochemical and photophysical studies were used to characterize designed redox-active copper sites. α3D, a de novo designed peptide that preassembles into a three-helix bundle fold, was functionalized with a triscysteine site to produce α3DIV. α3DIV structure was solved using Nuclear Magnetic Resonance (NMR). α3DIV comprised 1067 NMR restraints and 138 dihedral angles. The backbone of the 20 lowest energy structures has a root mean square deviation from the mean structure of 0.79 (0.16) Å, demonstrating a well-defined structure. The asymmetric 2HisCys(Met) copper electron transfer site, which is encompassed in the β-barrel fold of cupredoxins, was incorporated in α3D to examine whether the function and physical properties of cupredoxins can be recapitulated in an unrelated fold. This generated three designs: core, chelate and chelate-core constructs. Cu(II) binding to the core and chelate constructs displayed intense absorption bands between 380-400 nm (~2000 M−1 cm−1); the chelate-core construct showed two intense absorption bands at 401 (4429 M−1 cm−1) and 499 (2020 M−1 cm−1). X-ray absorption spectroscopy analysis on the Cu(I) adducts recapitulated the reduced state of cupredoxin proteins, producing short Cu-S(Cys) bonds at 2.16 – 2.23 Å. Overall, these results showed that the designed cupredoxin sites cannot enforce the structural constraints necessary for the appropriate Cu(II) chromophore, however the Cu(I) environment was retained. Moreover, the redox activity of the designed constructs was tested using electrochemical and photophysical methods. Electrochemical studies showed reduction potentials of +362 – +462 mV (vs. NHE), which are in the range of cupredoxins. Photophysical work revealed intermolecular ET activity with ruthenium(III)trisbipyridine produced first-order and bimolecular rate constants of 105 s−1 and 108 s−1 M−1, respectively. This work illustrates that the redox function of a native copper center in a β-barrel fold can be achieved in the α-helical framework of α3D. Further, the structure of α3DIV revealed a distorted triscysteine site, offering a model for proteins with thiol-rich ligands. Ultimately, this work provides a foundation for investigating long-range electron transfer reaction using de novo protein design.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113318/1/plegaria_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113318/2/plegaria_2.pd

Electron transfer activity of a de novo designed copper center in a three-helix bundle fold.

International audienceIn this work, we characterized the intermolecular electron transfer (ET) properties of a de novo designed metallopeptide using laser-flash photolysis. α3D-CH3 is three helix bundle peptide that was designed to contain a copper ET site that is found in the β-barrel fold of native cupredoxins. The ET activity of Cuα3D-CH3 was determined using five different photosensitizers. By exhibiting a complete depletion of the photo-oxidant and the successive formation of a Cu(II) species at 400 nm, the transient and generated spectra demonstrated an ET transfer reaction between the photo-oxidant and Cu(I)α3D-CH3. This observation illustrated our success in integrating an ET center within a de novo designed scaffold. From the kinetic traces at 400 nm, first-order and bimolecular rate constants of 10(5) s(-1) and 10(8) M(-1) s(-1) were derived. Moreover, a Marcus equation analysis on the rate versus driving force study produced a reorganization energy of 1.1 eV, demonstrating that the helical fold of α3D requires further structural optimization to efficiently perform ET. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson

De Novo Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins

International audienc

Intramolecular Photogeneration of a Tyrosine Radical in a Designed Protein

Long-distance biological electron transfer occurs through a hopping mechanism and often involves tyrosine as a high potential intermediate, for example in the early charge separation steps during photosynthesis. Protein design allows for the development of minimal systems to study the underlying principles of complex systems. Herein, we report the development of the first ruthenium-linked designed protein for the photogeneration of a tyrosine radical by intramolecular electron transfer

In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures

Bacterial microcompartments (BMCs) are organelles composed of a selectively permeable protein shell that encapsulates enzymes involved in CO2 fixation (carboxysomes) or carbon catabolism (metabolosomes). Confinement of sequential reactions by the BMC shell presumably increases the efficiency of the pathway by reducing the crosstalk of metabolites, release of toxic intermediates, and accumulation of inhibitory products. Because BMCs are composed entirely of protein and self-assemble, they are an emerging platform for engineering nanoreactors and molecular scaffolds. However, testing designs for assembly and function through in vivo expression is labor-intensive and has limited the potential of BMCs in bioengineering. Here, we developed a new method for in vitro assembly of defined nanoscale BMC architectures: shells and nanotubes. By inserting a "protecting group", a short ubiquitin-like modifier (SUMO) domain, self-assembly of shell proteins in vivo was thwarted, enabling preparation of concentrates of shell building blocks. Addition of the cognate protease removes the SUMO domain and subsequent mixing of the constituent shell proteins in vitro results in the self-assembly of three types of supramolecular architectures: a metabolosome shell, a carboxysome shell, and a BMC protein-based nanotube. We next applied our method to generate a metabolosome shell engineered with a hyper-basic luminal surface, allowing for the encapsulation of biotic or abiotic cargos functionalized with an acidic accessory group. This is the first demonstration of using charge complementarity to encapsulate diverse cargos in BMC shells. Collectively, our work provides a generally applicable method for in vitro assembly of natural and engineered BMC-based architectures

Protein Design: Toward Functional Metalloenzymes

The scope of this Review is to discuss the construction of metal sites in designed protein scaffolds. We categorize the effort of designing proteins into redesign, which is to rationally engineer desired functionality into an existing protein scaffold,(1-9) and de novo design, which is to build a peptidic or protein system that is not directly related to any sequence found in nature yet folds into a predicted structure and/or carries out desired reactions.(10-12) We will analyze and interpret the significance of designed protein systems from a coordination chemistry and biochemistry perspective, with an emphasis on those containing constructed metal sites as mimics for metalloenzymes