De Novo Designed Metallopeptides to Investigate Metal Ion Homeostasis, Electron Transfer, and Redox Catalysis.

Protein design is a powerful way to interrogate the basic requirements for function of metal sites by systematically incorporating elements important for function. Single-stranded three-helix bundles with either thiolate-rich sites for spectroscopic characterization and electron transfer, or histidine-rich sites for redox catalysis are described. Using a previous design, two constructs were designed to incorporate a fourth cysteine residue to investigate thiolate-rich sites involved in metal ion homeostasis and electron transfer. Rational re-design replaced a putative coordinating histidine with a cysteine. A second construct embedded a CXXC binding motif into the helical scaffold. These two constructs show different UV-visisble, 113Cd NMR, and 111mCd PAC, which indicate that they form different proportions of CdS3O and CdS4. The spectroscopy of these sites sheds light on how Cd(II) bindis to CadC and suggests a dynamic site in fast exchange with the solvent. Previous attempts at the design of a rubredoxin site have focused on reproducing the peptide fold around or using flexible loop regions to define the site in addition to canonical CXXC motifs. However, the use of CXXC motifs embedded in an α-helical scaffold produces a rubredoxin site that reproduces the Mössbauer, MCD, and EPR of rubredoxin without the use of loop regions. This successful design is the largest deviation from consensus rubredoxin and zinc finger folds reported. Electron transfer rates through a de novo designed scaffold were studied by the design and synthesis of a ruthenium trisbipyridine derivative appended to an exterior cysteine residues. A redox-active tyrosine in the 70th position is implicated as a relay amino acid from the iron center and absence of the tyrosine decreases the rate of electron transfer from the metal site. This is the first photo-generated tyrosine radical in a designed protein. A construct, which was previously reported for CO2 hydration, is substituted with copper and its spectroscopic and nitrite reductase activity are studied. This is the first demonstration of nitrite reductase activity in a single-stranded designed peptide. This thesis provides insight into designed proteins and their applications and lays the groundwork for further studies to progress towards a unified multifunctional redox protein.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113513/1/agtebo_1.pd

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity

Protein design is a useful strategy to interrogate the protein structureâ function relationship. We demonstrate using a highly modular 3â stranded coiled coil (TRIâ peptide system) that a functional typeâ 2 copper center exhibiting copper nitrite reductase (NiR) activity exhibits the highest homogeneous catalytic efficiency under aqueous conditions for the reduction of nitrite to NO and H2O. Modification of the amino acids in the second coordination sphere of the copper center increases the nitrite reductase activity up to 75â fold compared to previously reported systems. We find also that steric bulk can be used to enforce a threeâ coordinate CuI in a site, which tends toward twoâ coordination with decreased steric bulk. This study demonstrates the importance of the second coordination sphere environment both for controlling metalâ center ligation and enhancing the catalytic efficiency of metalloenzymes and their analogues.Erstklassiges aus der zweiten Reihe: Die Aktivität der Nitritreduktase kann durch Modifikation der sterischen Eigenschaften in der zweiten Koordinationssphäre eines Typâ 2â Kupferzentrums deutlich erhöht werden. à ber die Sterik lassen sich die Koordination und Reaktivität des Metalls in einem dreisträngigen â Coiledâ coilâ â TRIâ Peptidgerüst (TRIWâ H) vorgeben.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142882/1/ange201712757_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142882/2/ange201712757-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142882/3/ange201712757.pd

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity

Protein design is a useful strategy to interrogate the protein structureâ function relationship. We demonstrate using a highly modular 3â stranded coiled coil (TRIâ peptide system) that a functional typeâ 2 copper center exhibiting copper nitrite reductase (NiR) activity exhibits the highest homogeneous catalytic efficiency under aqueous conditions for the reduction of nitrite to NO and H2O. Modification of the amino acids in the second coordination sphere of the copper center increases the nitrite reductase activity up to 75â fold compared to previously reported systems. We find also that steric bulk can be used to enforce a threeâ coordinate CuI in a site, which tends toward twoâ coordination with decreased steric bulk. This study demonstrates the importance of the second coordination sphere environment both for controlling metalâ center ligation and enhancing the catalytic efficiency of metalloenzymes and their analogues.Second is best: A significant increase in nitrite reductase activity is achieved by modification of the steric properties of the second coordination sphere of a typeâ 2 copper center. The steric properties can be harnessed to control metal coordination and reactivity in a 3â stranded coiled coil TRI peptide scaffold (TRIWâ H).Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142897/1/anie201712757.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142897/2/anie201712757-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142897/3/anie201712757_am.pd

Fluorogenic Labeling Strategies for Biological Imaging

The spatiotemporal fluorescence imaging of biological processes requires effective tools to label intracellular biomolecules in living systems. This review presents a brief overview of recent labeling strategies that permits one to make protein and RNA strongly fluorescent using synthetic fluorogenic probes. Genetically encoded tags selectively binding the exogenously applied molecules ensure high labeling selectivity, while high imaging contrast is achieved using fluorogenic chromophores that are fluorescent only when bound to their cognate tag, and are otherwise dark. Beyond avoiding the need for removal of unbound synthetic dyes, these approaches allow the development of sophisticated imaging assays, and open exciting prospects for advanced imaging, particularly for multiplexed imaging and super-resolution microscopy

Variable primary coordination environments of Cd(ɪɪ) binding to three helix bundles provide a pathway for rapid metal exchange

Members of the ArsR/SmtB family of transcriptional repressors, such as CadC, regulate the intracellular levels of heavy metals like Cd(II), Hg(II), and Pb(II). These metal sensing proteins bind their target metals with high specificity and affinity, however, a lack of structural information about these proteins makes defining the coordination sphere of the target metal difficult. Lingering questions as to the identity of Cd(II) coordination in CadC are addressed via protein design techniques. Two designed peptides with tetrathiolate metal binding sites were prepared and characterized, revealing fast exchange between CdS(3)O and CdS(4) coordination spheres. Correlation of (111m)Cd PAC spectroscopy and (113)Cd NMR spectroscopy suggests that Cd(II) coordinated to CadC is in fast exchange between CdS(3)O and CdS(4) forms, which may provide a mechanism for rapid sensing of heavy metal contaminants by this regulatory protein

A far-red fluorescent chemogenetic reporter for in vivo molecular imaging

International audienceFar-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially nonfluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae and chicken embryo. Beyond enabling genetic encoding of far-red fluorescence, frFAST allowed the design of a farred chemogenetic reporter of protein-protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors

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