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Catalysis and Electron Transfer in De Novo Designed Helical Scaffolds.

ABSTRACT: The relationship between protein structure and function is one of the greatest puzzles within biochemistry. De novo metalloprotein design is a way to wipe the board clean and determine what is required to build in function from the ground up in an unrelated structure. This Review focuses on protein design efforts to create de novo metalloproteins within alpha-helical scaffolds. Examples of successful designs include those with carbonic anhydrase or nitrite reductase activity by incorporating a ZnHis₃ or CuHis₃ site, or that recapitulate the spectroscopic properties of unique electron-transfer sites in cupredoxins (CuHis₂ Cys) or rubredoxins (FeCys₄ ). This work showcases the versatility of alpha helices as scaffolds for metalloprotein design and the progress that is possible through careful rational design. Our studies cover the invariance of carbonic anhydrase activity with different site positions and scaffolds, refinement of our cupredoxin models, and enhancement of nitrite reductase activity up to 1000-fold.

SUBMITTER: Pinter TBJ

PROVIDER: S-EPMC7035182 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Catalysis and Electron Transfer in <i>De Novo</i> Designed Metalloproteins.

Project description:One of the hallmark advances in our understanding of metalloprotein function is showcased in our ability to design new, non-native, catalytically active protein scaffolds. This review highlights progress and milestone achievements in the field of de novo metalloprotein design focused on reports from the past decade with special emphasis on de novo designs couched within common subfields of bioinorganic study: heme binding proteins, monometal- and dimetal-containing catalytic sites, and metal-containing electron transfer sites. Within each subfield, we highlight several of what we have identified as significant and important contributions to either our understanding of that subfield or de novo metalloprotein design as a discipline. These reports are placed in context both historically and scientifically. General suggestions for future directions that we feel will be important to advance our understanding or accelerate discovery are discussed.

| S-EPMC10735231 | biostudies-literature

Control of proton and electron transfer in de novo designed, biomimetic ? hairpins.

Project description:Tyrosine side chains are involved in proton coupled electron transfer reactions (PCET) in many complex proteins, including photosystem II (PSII) and ribonucleotide reductase. For example, PSII contains two redox-active tyrosines, TyrD (Y160D2) and TyrZ (Y161D1), which have different protein environments, midpoint potentials, and roles in catalysis. TyrD has a midpoint potential lower than that of TyrZ, and its protein environment is distinguished by potential ?-cation interactions with arginine residues. Designed biomimetic peptides provide a system that can be used to investigate how the protein matrix controls PCET reactions. As a model for the redox-active tyrosines in PSII, we are employing a designed, 18 amino acid ? hairpin peptide in which PCET reactions occur between a tyrosine (Tyr5) and a cross-strand histidine (His14). In this peptide, the single tyrosine is hydrogen-bonded to an arginine residue, Arg16, and a second arginine, Arg12, has a ?-cation interaction with Tyr5. In this report, the effect of these hydrogen bonding and electrostatic interactions on the PCET reactions is investigated. Electrochemical titrations show that histidine substitutions change the nature of PCET reactions, and optical titrations show that Arg16 substitution changes the pK of Tyr5. Removal of Arg16 or Arg12 increases the midpoint potential for tyrosine oxidation. The effects of Arg12 substitution are consistent with the midpoint potential difference, which is observed for the PSII redox-active tyrosine residues. Our results demonstrate that a ?-cation interaction, hydrogen bonding, and PCET reactions alter redox-active tyrosine function. These interactions can contribute equally to the control of midpoint potential and reaction rate.

| S-EPMC3042127 | biostudies-literature

Minimal Heterochiral de Novo Designed 4Fe-4S Binding Peptide Capable of Robust Electron Transfer.

Project description:Ambidoxin is a designed, minimal dodecapeptide consisting of alternating L and D amino acids that binds a 4Fe-4S cluster through ligand-metal interactions and an extensive network of second-shell hydrogen bonds. The peptide can withstand hundreds of oxidation-reduction cycles at room temperature. Ambidoxin suggests how simple, prebiotic peptides may have achieved robust redox catalysis on the early Earth.

| S-EPMC7050467 | biostudies-literature

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

Project description:In this work, we characterized the intermolecular electron transfer (ET) properties of a de novo designed metallopeptide using laser-flash photolysis. ?3D-CH3 is three helix bundle peptide that was designed to contain a copper ET site that is found in the ?-barrel fold of native cupredoxins. The ET activity of Cu?3D-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)?3D-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 ?3D 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.

| S-EPMC5233711 | biostudies-literature

Photoinduced Electron Transfer Elicits a Change in the Static Dielectric Constant of a de Novo Designed Protein.

Project description:We provide a direct measure of the change in effective dielectric constant (?(S)) within a protein matrix after a photoinduced electron transfer (ET) reaction. A linked donor-bridge-acceptor molecule, PZn-Ph-NDI, consisting of a (porphinato)Zn donor (PZn), a phenyl bridge (Ph), and a naphthalene diimide acceptor (NDI), is shown to be a "meter" to indicate protein dielectric environment. We calibrated PZn-Ph-NDI ET dynamics as a function of solvent dielectric, and computationally de novo designed a protein SCPZnI3 to bind PZn-Ph-NDI in its interior. Mapping the protein ET dynamics onto the calibrated ET catalogue shows that SCPZnI3 undergoes a switch in the effective dielectric constant following photoinduced ET, from ?(S) ? 8 to ?(S) ? 3.

| S-EPMC5049705 | biostudies-literature

Anti-plasmodial action of de novo-designed, cationic, lysine-branched, amphipathic, helical peptides.

Project description:BackgroundA lack of vaccine and rampant drug resistance demands new anti-malarials.MethodsIn vitro blood stage anti-plasmodial properties of several de novo-designed, chemically synthesized, cationic, amphipathic, helical, antibiotic peptides were examined against Plasmodium falciparum using SYBR Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy and FACS analysis.ResultsUnlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH2) (X?=?F,?F) (Fm, ?Fm IC50 >100 ?M)}, the lysine-branched,dimeric versions showed far greater potency {IC50 (?M) Fd 1.5 , ?Fd 1.39}. The more helical and proteolytically stable ?Fd was studied for mechanistic details. ?Fq, a K-K2 dendrimer of ?Fm and (?Fm)2 a linear dimer of ?Fm showed IC50 (?M) of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity indices were >35 (?Fd), >20 (?Fm)2 and 10 (?Fq). FITC-?Fd showed rapid and selective accumulation in parasitized red cells. Overlaying DAPI and FITC florescence suggested that ?Fd binds DNA. Trophozoites and schizonts incubated with ?Fd (2.5??M) egressed anomalously and Band-3 immunostaining revealed them not to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were found to be invasion incompetent.ConclusionGood selectivity (>35), good resistance index (1.1) and low cytotoxicity indicate the promise of ?Fd against malaria.

| S-EPMC3502156 | biostudies-literature

Segmentation strategy of de novo designed four-helical bundles expands protein oligomerization modalities for cell regulation.

Project description:Protein-protein interactions govern most biological processes. New protein assemblies can be introduced through the fusion of selected proteins with di/oligomerization domains, which interact specifically with their partners but not with other cellular proteins. While four-helical bundle proteins (4HB) have typically been assembled from two segments, each comprising two helices, here we show that they can be efficiently segmented in various ways, expanding the number of combinations generated from a single 4HB. We implement a segmentation strategy of 4HB to design two-, three-, or four-chain combinations for the recruitment of multiple protein components. Different segmentations provide new insight into the role of individual helices for 4HB assembly. We evaluate 4HB segmentations for potential use in mammalian cells for the reconstitution of a protein reporter, transcriptional activation, and inducible 4HB assembly. Furthermore, the implementation of trimerization is demonstrated as a modular chimeric antigen receptor for the recognition of multiple cancer antigens.

| S-EPMC10082849 | biostudies-literature

Designed for life: biocompatible de novo designed proteins and components.

Project description:A principal goal of synthetic biology is the de novo design or redesign of biomolecular components. In addition to revealing fundamentally important information regarding natural biomolecular engineering and biochemistry, functional building blocks will ultimately be provided for applications including the manufacture of valuable products and therapeutics. To fully realize this ambitious goal, the designed components must be biocompatible, working in concert with natural biochemical processes and pathways, while not adversely affecting cellular function. For example, de novo protein design has provided us with a wide repertoire of structures and functions, including those that can be assembled and function in vivo Here we discuss such biocompatible designs, as well as others that have the potential to become biocompatible, including non-protein molecules, and routes to achieving full biological integration.

| S-EPMC6127164 | biostudies-literature

A de novo designed protein protein interface.

Project description:As an approach to both explore the physical/chemical parameters that drive molecular self-assembly and to generate novel protein oligomers, we have developed a procedure to generate protein dimers from monomeric proteins using computational protein docking and amino acid sequence design. A fast Fourier transform-based docking algorithm was used to generate a model for a dimeric version of the 56-amino-acid beta1 domain of streptococcal protein G. Computational amino acid sequence design of 24 residues at the dimer interface resulted in a heterodimer comprised of 12-fold and eightfold variants of the wild-type protein. The designed proteins were expressed, purified, and characterized using analytical ultracentrifugation and heteronuclear NMR techniques. Although the measured dissociation constant was modest ( approximately 300 microM), 2D-[(1)H,(15)N]-HSQC NMR spectra of one of the designed proteins in the absence and presence of its binding partner showed clear evidence of specific dimer formation.

| S-EPMC2222823 | biostudies-literature

De novo designed cyclic-peptide heme complexes.

Project description:The structural characterization of de novo designed metalloproteins together with determination of chemical reactivity can provide a detailed understanding of the relationship between protein structure and functional properties. Toward this goal, we have prepared a series of cyclic peptides that bind to water-soluble metalloporphyrins (FeIII and CoIII). Neutral and positively charged histidine-containing peptides bind with a high affinity, whereas anionic peptides bind only weakly to the negatively charged metalloporphyrin. Additionally, it was found that the peptide becomes helical only in the presence of the metalloporphyrin. CD experiments confirm that the metalloporphyrin binds specific cyclic peptides with high affinity and with isodichroic behavior. Thermal unfolding experiments show that the complex has "native-like" properties. Finally, NMR spectroscopy produced well dispersed spectra and experimental restraints that provide a high-resolution solution structure of the complexed peptide.

| S-EPMC263730 | biostudies-literature

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