9 research outputs found
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 ÎČ-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
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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
<i>De Novo</i> Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins
Using <i>de novo</i> protein design, we incorporated a copper metal binding
site within the three-helix bundle α<sub>3</sub>D (Walsh et
al.<i> Proc. Natl. Acad. Sci. U.S.A.</i> <b>1999</b>, 96, 5486â5491) to assess whether a cupredoxin center within
an α-helical domain could mimic the spectroscopic, structural,
and redox features of native type-1 copper (CuT1) proteins. We aimed
to determine whether a CuT1 center could be realized in a markedly
different scaffold rather than the native ÎČ-barrel fold and
whether the characteristic short CuâS bond (2.1â2.2
Ă
) and positive reduction potentials could be decoupled from
the spectroscopic properties (Δ<sub>600 nm</sub> = 5000
M<sup><b>â</b>1</sup> cm<sup><b>â</b>1</sup>) of such centers. We incorporated 2HisCysÂ(Met) residues in three
distinct α<sub>3</sub>D designs designated core (CR), chelate
(CH), and chelate-core (ChC). XAS analysis revealed a coordination
environment similar to reduced CuT1 proteins, producing CuâSÂ(Cys)
bonds ranging from 2.16 to 2.23 Ă
and CuâNÂ(His) bond distances
of 1.92â1.99 Ă
. However, CuÂ(II) binding to the CR and
CH constructs resulted in tetragonal type-2 copper-like species, displaying
an intense absorption band between 380 and 400 nm (>1500 M<sup><b>â</b>1</sup> cm<sup><b>â</b>1</sup>)
and <i>A</i><sub>||</sub> values of (150â185) Ă
10<sup><b>â</b>4</sup> cm<sup><b>â</b>4</sup>. The ChC construct, which possesses a metal-binding site deeper
in its helical bundle, yielded a CuT1-like brown copper species, with
two absorption bands at 401 (4429 M<sup><b>â</b>1</sup> cm<sup><b>â</b>1</sup>) and 499 (2020 M<sup><b>â</b>1</sup> cm<sup><b>â</b>1</sup>) nm and an <i>A</i><sub>||</sub> value âŒ30 Ă 10<sup>â4</sup> cm<sup>â4</sup> greater than its native counterparts. Electrochemical
studies demonstrated reduction potentials of +360 to +460 mV (vs NHE),
which are within the observed range for azurin and plastocyanin. These
observations showed that the designed metal binding sites lacked the
necessary rigidity to enforce the appropriate structural constraints
for a CuÂ(II) chromophore (EPR and UVâvis); however, the CuÂ(I)
structural environment and the high positive potential of CuT1 centers
were recapitulated within the α-helical bundle of α<sub>3</sub>D
Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster
Bacterial microcompartments (BMCs)
are self-assembling organelles
composed of a selectively permeable protein shell and encapsulated
enzymes. They are considered promising templates for the engineering
of designed bionanoreactors for biotechnology. In particular, encapsulation
of oxidoreductive reactions requiring electron transfer between the
lumen of the BMC and the cytosol relies on the ability to conduct
electrons across the shell. We determined the crystal structure of
a component protein of a synthetic BMC shell, which informed the rational
design of a [4Fe-4S] cluster-binding site in its pore. We also solved
the structure of the [4Fe-4S] cluster-bound, engineered protein to
1.8 Ă
resolution, providing the first structure of a BMC shell
protein containing a metal center. The [4Fe-4S] cluster was characterized
by optical and EPR spectroscopies; it has a reduction potential of
â370 mV vs the standard hydrogen electrode (SHE) and is stable
through redox cycling. This remarkable stability may be attributable
to the hydrogen-bonding network provided by the main chain of the
protein scaffold. The properties of the [4Fe-4S] cluster resemble
those in low-potential bacterial ferredoxins, while its ligation to
three cysteine residues is reminiscent of enzymes such as aconitase
and radical <i>S</i>-adenosymethionine (SAM) enzymes. This
engineered shell protein provides the foundation for conferring electron-transfer
functionality to BMC shells
Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster
Bacterial microcompartments (BMCs)
are self-assembling organelles
composed of a selectively permeable protein shell and encapsulated
enzymes. They are considered promising templates for the engineering
of designed bionanoreactors for biotechnology. In particular, encapsulation
of oxidoreductive reactions requiring electron transfer between the
lumen of the BMC and the cytosol relies on the ability to conduct
electrons across the shell. We determined the crystal structure of
a component protein of a synthetic BMC shell, which informed the rational
design of a [4Fe-4S] cluster-binding site in its pore. We also solved
the structure of the [4Fe-4S] cluster-bound, engineered protein to
1.8 Ă
resolution, providing the first structure of a BMC shell
protein containing a metal center. The [4Fe-4S] cluster was characterized
by optical and EPR spectroscopies; it has a reduction potential of
â370 mV vs the standard hydrogen electrode (SHE) and is stable
through redox cycling. This remarkable stability may be attributable
to the hydrogen-bonding network provided by the main chain of the
protein scaffold. The properties of the [4Fe-4S] cluster resemble
those in low-potential bacterial ferredoxins, while its ligation to
three cysteine residues is reminiscent of enzymes such as aconitase
and radical <i>S</i>-adenosymethionine (SAM) enzymes. This
engineered shell protein provides the foundation for conferring electron-transfer
functionality to BMC shells
Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster
Bacterial microcompartments (BMCs)
are self-assembling organelles
composed of a selectively permeable protein shell and encapsulated
enzymes. They are considered promising templates for the engineering
of designed bionanoreactors for biotechnology. In particular, encapsulation
of oxidoreductive reactions requiring electron transfer between the
lumen of the BMC and the cytosol relies on the ability to conduct
electrons across the shell. We determined the crystal structure of
a component protein of a synthetic BMC shell, which informed the rational
design of a [4Fe-4S] cluster-binding site in its pore. We also solved
the structure of the [4Fe-4S] cluster-bound, engineered protein to
1.8 Ă
resolution, providing the first structure of a BMC shell
protein containing a metal center. The [4Fe-4S] cluster was characterized
by optical and EPR spectroscopies; it has a reduction potential of
â370 mV vs the standard hydrogen electrode (SHE) and is stable
through redox cycling. This remarkable stability may be attributable
to the hydrogen-bonding network provided by the main chain of the
protein scaffold. The properties of the [4Fe-4S] cluster resemble
those in low-potential bacterial ferredoxins, while its ligation to
three cysteine residues is reminiscent of enzymes such as aconitase
and radical <i>S</i>-adenosymethionine (SAM) enzymes. This
engineered shell protein provides the foundation for conferring electron-transfer
functionality to BMC shells
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