► De novo metalloprotein design. ► Structural metal sites: Hg(II) binding to α-helical coiled coils. ► Hydrolytic catalysis by a de novo designed Zn(II) metalloprotein. ► Redox catalysis by a de novo ...designed Cu(I/II) metalloprotein. ► De novo designed binuclear metal sites.
Metalloenzymes efficiently catalyze some of the most important and difficult reactions in nature. For many years, coordination chemists have effectively used small molecule models to understand these systems. More recently, protein design has been shown to be an effective approach for mimicking metal coordination environments. Since the first designed proteins were reported, much success has been seen for incorporating metal sites into proteins and attaining the desired coordination environment but until recently, this has been with a lack of significant catalytic activity. Now there are examples of designed metalloproteins that, although not yet reaching the activity of native enzymes, are considerably closer. In this review, we highlight work leading up to the design of a small metalloprotein containing two metal sites, one for structural stability (HgS3) and the other a separate catalytic zinc site to mimic carbonic anhydrase activity (ZnN3O). The first section will describe previous studies that allowed for a high affinity thiolate site that binds heavy metals in a way that stabilizes three-stranded coiled coils. The second section will examine ways of preparing histidine-rich environments that lead to metal-based hydrolytic catalysts. We will also discuss other recent examples of the design of structural metal sites and functional metalloenzymes. Our work demonstrates that attaining the proper first coordination geometry of a metal site can lead to a significant fraction of catalytic activity, apparently independent of the type of secondary structure of the surrounding protein environment. We are now in a position to begin to meet the challenge of building a metalloenzyme systematically from the bottom-up by engineering and analyzing interactions directly around the metal site and beyond.
Zinc is an essential element required for the function of more than 300 enzymes spanning all classes. Despite years of dedicated study, questions regarding the connections between primary and ...secondary metal ligands and protein structure and function remain unanswered, despite numerous mechanistic, structural, biochemical, and synthetic model studies. Protein design is a powerful strategy for reproducing native metal sites that may be applied to answering some of these questions and subsequently generating novel zinc enzymes. From examination of the earliest design studies introducing simple Zn(II)-binding sites into de novo and natural protein scaffolds to current studies involving the preparation of efficient hydrolytic zinc sites, it is increasingly likely that protein design will achieve reaction rates previously thought possible only for native enzymes. This Current Topic will review the design and redesign of Zn(II)-binding sites in de novo-designed proteins and native protein scaffolds toward the preparation of catalytic hydrolytic sites. After discussing the preparation of Zn(II)-binding sites in various scaffolds, we will describe relevant examples for reengineering existing zinc sites to generate new or altered catalytic activities. Then, we will describe our work on the preparation of a de novo-designed hydrolytic zinc site in detail and present comparisons to related designed zinc sites. Collectively, these studies demonstrate the significant progress being made toward building zinc metalloenzymes from the bottom up.
Recent advances in the study of the oxygen evolving complex (OEC) of photosystem II (PSII) include structural information attained from several X-ray crystallographic (XRD) and spectroscopic (XANES ...and EXAFS) investigations. The possible structural features gleaned from these studies have enabled synthetic chemists to design more accurate model complexes, which in turn, offer better insight into the possible pathways used by PSII to drive photosynthetic water oxidation catalysis. Mononuclear model compounds have been used to advance the knowledge base regarding the physical properties and reactivity of high-valent (Mn
IV or Mn
V) complexes. Such investigations have been especially important in regard to the manganyl (Mn
IV
O or Mn
V(O) species, as there are no reports, to date, of any structurally characterized multinuclear model compounds that incorporate such a functionality. Dinuclear and trinuclear model compounds have also been thoroughly studied in attempts to draw further comparison to the physical properties observed in the natural system and to design systems of catalytic relevance. As the reactive center of the OEC has been shown to contain an oxo-Mn
4Ca cluster, exact structural models necessitate a tetranuclear Mn core. The number of models that make use of Mn
4 clusters has risen substantially in recent years, and these models have provided evidence to support and refute certain mechanistic proposals. Further work is needed to adequately address the rationale for Ca (and Cl) in the OEC and to determine the sequence of events that lead to O
2 evolution.
Sensitive detection of cell necrosis is crucial for the determination of cell viability. Because of its high resolution at the cellular level and sensitivity, optical imaging is highly attractive for ...identifying cell necrosis. However, challenges associated with this technique remain present such as the rapid photobleaching of several types of organic fluorophores and/or the interference generated by biological autofluorescence. Herein, we synthesized novel biologically compatible Zn2+/Ln3+ metallacrowns (MCs) that possess attractive near-infrared (NIR) emission and are highly photostable. In addition, these MCs have the ability to label differentially necrotic HeLa cells from living cells. This work is also the first demonstration of (i) the use of the NIR emission arising from a single lanthanide(III) cation for optical biological imaging of cells under single photon excitation, (ii) the first example of a lanthanide(III)-based NIR-emitting probe that can be targeted to a specific type of cell.
Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge ...to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions-a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO(2) hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO(2) hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.
Conspectus The relationship between structure and function has long been one of the major points of investigation in Biophysics. Understanding how much, or how little, of a protein’s often ...complicated structure is necessary for its function can lead to directed therapeutic strategies and would allow one to design proteins for specific desired functions. Studying protein function by de novo design builds the functionality from the ground up in a completely unrelated and noncoded protein scaffold. Our lab has used this strategy to study heavy and transition metal binding within the TRI family of three stranded coiled coil (3SCC) constructs to understand coordination geometry and metalloenzyme catalytic control within a protein environment. These peptides contain hydrophobic layers within the interior of the 3SCC, which one can mutate to metal binding residues to create a minimal metal binding site, while solid phase synthesis allows our lab to easily incorporate a number of noncoded amino acids including d enantiomers of binding or secondary coordination sphere amino acids, penicillamine, or methylated versions of histidine. Our studies of Cd(II) binding to Cys3 environments have determined, largely through the use of 113Cd NMR and 111mCd PAC, that the coordination environment around a heavy metal can be controlled by incorporating noncoded amino acids in either the primary or secondary coordination spheres. We found mutating the metal binding amino acids to l-Pen can enforce trigonal Cd(II)S3 geometry exclusively compared to the mixed coordination determined for l-Cys coordination. The same result can be achieved with secondary sphere mutations as well by incorporating d-Leu above a Cys3. We hypothesize this latter effect is due to the increased steric packing above the metal binding site that occurs when the l-Leu oriented toward the N-terminus of the scaffold is mutated to d-Leu and oriented toward the C-terminus. Mutating the layer below Cys3 to d-Leu instead formed a mixed 4- and 5-coordinate Cd(II)S3(H2O) and Cd(II)S3(H2O)2 construct as steric bulk was decreased below the metal binding site. We have also applied noncoded amino acids to metalloenzyme systems by incorporating His residues that are methylated at the δ- or ε-nitrogen to enforce Cu(I) ligation to the opposite open nitrogen of His and found a 2 orders of magnitude increased catalytic efficiency for nitrite reductase activity with ε-nitrogen coordination compared to δ-nitrogen. These results exemplify the ability to tune coordination environment and catalytic efficiency within a de novo scaffold as well as the utility of noncoded amino acids to increase the chemist’s toolbox. By furthering our understanding of metalloprotein design one could envision, through our use of amino acids not normally available to nature, that protein design laboratories will soon be capable of outperforming the native systems previously used as their benchmark of successful design. The ability to design proteins at this level would have far reaching and exciting benefits within various fields including medical and industrial applications.
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.
Two series of lanthanide complexes have been chosen to analyze trends in the magnetic properties and crystal field parameters (CFPs) along the two series: The highly symmetric LnZn16(picHA)16 series ...(Ln=Tb, Dy, Ho, Er, Yb; picHA=picolinohydroxamic acid) and the Ln(dpa)3(C3H5N2)3⋅3H2O series (Ln=Ce–Yb; dpa=2,6‐dipicolinic acid) with approximate three‐fold symmetry. The first series presents a compressed coordination sphere of eight oxygen atoms whereas in the second series, the coordination sphere consists of an elongated coordination sphere formed of six oxygen atoms. The CFPs have been deduced from ab initio calculations using two methods: The AILFT (ab initio ligand field theory) method, in which the parameters are determined at the orbital level, and the ITO (irreducible tensor operator) decomposition, in which the problems are treated at the many‐electron level. It has been found that the CFPs are transferable from one derivative to another, within a given series, as a first approximation. The sign of the second‐order parameter B02
differs in the two series, reflecting the different environments. It has been found that the use of the strength parameter S allows for an easy comparison between complexes. Furthermore, in both series, the parameters have been found to decrease in magnitude along the series, and this decrease is attributed to covalent effects.
The magnetic properties and crystal field parameters (CFPs) across two series of lanthanide complexes have been explored by ab initio calculations. It has been found that the CFPs are transferable from one derivative to another, within a given series, as a first approximation. Furthermore, in both series, the parameters have been found to decrease in magnitude across the series, attributed to covalent effects.
White light production is of major importance for ambient lighting and technological displays. White light can be obtained by several types of materials and their combinations, but single component ...emitters remain rare and desirable towards thinner devices that are, therefore, easier to control and that require fewer manufacturing steps. We have designed a series of dysprosium(iii)-based luminescent metallacrowns (MCs) to achieve this goal. The synthesized MCs possess three main structural types LnGa
(L')
(L'')
(type A), Ln
Ga
(L')
(L''')
(type B) and LnGa
(L')
(OH)
(type C) (H
L', HL'' and H
L''' derivatives of salicylhydroxamic, benzoic and isophthalic acids, respectively). The advantage of these MCs is that, within each structural type, the nature of the organic building blocks does not affect the symmetry around Dy
. By detailed studies of the photophysical properties of these Dy
-based MCs, we have demonstrated that CIE coordinates can be tuned from warm to neutral to cold white by (i) defining the symmetry about Dy
, and (ii) choosing appropriate chromophoric building blocks. These organic building blocks, without altering the coordination geometry around Dy
, influence the total emission profile through changing the probability of different energy transfer processes including the
T
← Dy
* energy back transfer and/or by generating ligand-centered fluorescence in the blue range. This work opens new perspectives for the creation of white light emitting devices using single component tetrachroic molecular compounds.
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