46 research outputs found
Protein Design: Toward Functional Metalloenzymes
No full textThe 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
De Novo Designed Metallopeptides to Investigate Metal Ion Homeostasis, Electron Transfer, and Redox Catalysis.
No full textProtein 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
Sensing cellular biochemistry with fluorescent chemical-genetic hybrids
No full textInternational audienceFluorescent biosensors are powerful tools with which to detect biochemical events inside of cells with high spatiotemporal resolution. Biosensors based on fluorescent proteins often suffer from issues with photostability and brightness. On the other hand, hybrid, chemical-genetic systems present unique opportunities to combine the strengths of synthetic, organic chemistry with biological macromolecules to generate exquisitely tailored semisynthetic sensors
Structure-Function Dataset Reveals Environment Effects within a Fluorescent Protein Model System
No full textAnisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure-function relationships. We examined this using fluorescent proteins as easy-to-modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.sponsorship: We are grateful to Gerrit Groenhof (University of Jyvaskyla), Jeremy Harvey (KU Leuven), Raffaele Vitale (Universite de Lille), and Dominique Bourgeois (Institut de Biologie Structurale) for critical insights and discussion. E.D.Z. and L.V.M. thank the beamline staff from X06DA at the Swiss Light Source (Villigen, Switzerland), Proxima1 and Proxima2A at synchrotron Soleil (Gif-sur-Yvette, France), XRD1 at Elettra (Trieste, Italy) and I03 at Diamond Light Source (Oxfordshire, UK) for assistance during X-ray diffraction data collection. E.D.Z., S.H., and S.D. thank the Research Foundation Flanders (FWO) for a doctoral fellowship and postdoctoral fellowships (12X7919N and 12R2817N). This work was supported through funding from the Research Foundation Flanders through grants 1514319 N, G090819N, G0B8817N, and the European Research Council through grant 714688 NanoCellActivity. (Research Foundation Flanders (FWO)|12X7919N, Research Foundation Flanders (FWO)|12R2817N, Research Foundation Flanders|1514319 N, Research Foundation Flanders|G090819N, Research Foundation Flanders|G0B8817N, European Research Council|714688)status: Publishe
Fluorogenic Labeling Strategies for Biological Imaging
No full textInternational audienceThe 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
Orthogonal fluorescent chemogenetic reporters for multicolor imaging
No full textInternational audienceSpectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein–protein interactions by live-cell fluorescence microscopy
Variable primary coordination environments of Cd(ɪɪ) binding to three helix bundles provide a pathway for rapid metal exchange
No full textMembers 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 CdS3O and CdS4 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 CdS3O and CdS4 forms, which may provide a mechanism for rapid sensing of heavy metal contaminants by this regulatory protein.</p
A Far‐Red Emitting Fluorescent Chemogenetic Reporter for In Vivo Molecular Imaging
No full textInternational 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
Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy
No full textInducible chemical-genetic fluorescent
markers are promising tools
for live cell imaging requiring high spatiotemporal resolution and
low background fluorescence. The fluorescence-activating and absorption
shifting tag (FAST) was recently developed to form fluorescent molecular
complexes with a family of small, synthetic fluorogenic chromophores
(so-called fluorogens). Here, we use rational design to modify the
binding pocket of the protein and screen for improved fluorescence
performances with four different fluorogens. The introduction of a
single mutation results in improvements in both quantum yield and
dissociation constant with nearly all fluorogens tested. Our improved
FAST (iFAST) allowed the generation of a tandem iFAST (td-iFAST) that
forms green and red fluorescent reporters 1.6-fold and 2-fold brighter
than EGFP and mCherry, respectively, while having a comparable size
