Siu Yee New

Design of a single protein that spans the entire 2-V range of physiological redox potentials

Significance Nature spent millions of years to evolve electron transfer proteins that span a wide range of reduction potentials (E°′) under physiological conditions, from −1 V to 1 V vs. standard hydrogen electrode. Understanding the rules that govern such tuning within similar classes of metalloproteins enables scientists to rationally tune the E°′of their catalysts without changing the active site. An ultimate test of such understanding is to achieve the entire range of E°′ within a single protein, a feat that has not been achieved yet. Herein, we conclusively found that we can cover the entire 2-V range of E°′ using a single protein with five mutations and two metal ions. We have also provided explanations for structural features responsible for such high potential. The reduction potential (E°′) is a critical parameter in determining the efficiency of most biological and chemical reactions. Biology employs three classes of metalloproteins to cover the majority of the 2-V range of physiological E°′s. An ultimate test of our understanding of E°′ is to find out the minimal number of proteins and their variants that can cover this entire range and the structural features responsible for the extreme E°′. We report herein the design of the protein azurin to cover a range from +970 mV to −954 mV vs. standard hydrogen electrode (SHE) by mutating only five residues and using two metal ions. Spectroscopic methods have revealed geometric parameters important for the high E°′. The knowledge gained and the resulting water-soluble redox agents with predictable E°′s, in the same scaffold with the same surface properties, will find wide applications in chemical, biochemical, biophysical, and biotechnological fields.

DNA-templated silver nanoclusters: structural correlation and fluorescence modulation

12 years after the introduction of DNA-templated silver nanoclusters (DNA-AgNCs), exciting progress has been made and yet we are still in the midst of trying to fully understand this nanomaterial. The prominent excellence of DNA-AgNCs is undoubtedly its modulatable emission property, of which how variation in DNA templates causes emission tuning remains elusive. Based on the up-to-date DNA-AgNCs, we aim to establish the correlation between the structure/sequence of DNA templates and emission behaviour of AgNCs. Herein, we systematically present a wide-range of DNA-AgNCs based on the structural complexity of the DNA templates, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), triple-stranded DNA (tsDNA) and DNA nanostructures. For each DNA category, we discuss the emission property, quantum yield and synthesis condition of the respective AgNCs, before cross-comparing the impact of different DNA scaffolds on the properties of AgNCs. A future outlook for this area is given as a conclusion. By putting the information together, this review may shed new light on understanding DNA-AgNCs while we are expecting continuous breakthroughs in this field.

Redox tuning of two biological copper centers through non-covalent interactions: same trend but different magnitude

The same non-covalent interactions previously found to affect the redox potential (E(m)) of the mononuclear T1 Cu protein azurin (Az) are shown to also fine-tune the E(m) of the dinuclear Cu(A) center in the same Az protein scaffold. The effects of these mutations are in the same direction but with smaller magnitude in the Cu(A) site, due to dissipation of the effects by the dinuclear Cu(A) center.

Supramolecular assembly of a new series of copper-L-arginine Schiff bases

A series of copper-L-arginine complexes has been synthesized and characterized by spectroscopic and single-crystal X-ray diffraction analyses. The L-arginine Schiff bases invariably show [ONO] tridentate coordination on a mononuclear Cu(II) sphere. [Cu(RNap)(OAc)]·2H2O, 1 (RNap = N-(2-hydroxy-1-naphthalidene) L-arginine), obtained from Cu(OAc)2·2H2O and HRNap, gives two stereochemically different molecules in the crystal unit cell, with each of them showing different acetate binding modes. It exhibits a channel structure with disordered water molecules filling the cavity. Complexation of N-(2-hydroxy-5-nitro-1-salicylidene) L-arginine (HRNO2) with Cu(ClO4)2·6H2O yields the intriguing triclinic crystal structure of 2. Its unit cell consists of a neutral molecule [Cu(RNO2)Cl(H2O)], an ion pair [Cu(RNO2)(H2O)2]ClO4 and three water molecules of crystallization. Complexation of HRMe (RMe = N-(2-hydroxy-5-methyl-1-salicylidene) with Cu(OAc)2·2H2O gives [Cu(RMe)(OAc)]·5H2O, 3, whose crystal packing shows a zeolite-like network with a water cluster residing in the hydrophilic channel, thus facilitating the hydrogen bonding interaction.

Lipid-bilayer-mimicking solid-state structures of Cu(II) and Ni(II) with L-tryptophan and L-tyrosine Schiff base derivatives

New metal-amino acid Schiff bases resembling lipid bilayers have been isolated and established by single-crystal X-ray crystallography. Hydrogen bonding between the solvate and coordination sphere and aromatic stacking of the Schiff base ligands lead to discrete hydrophilic and hydrophobic regions. [Cu2(WNap)2(H2O)2]·2H2O, 1 (WNap = N-(2-hydroxy-1-naphthalidene)-L-tryptophan), gives bilayer sheets with width ratio of ca. 3 A : 5 A (hydrophilic : hydrophobic). Replacement of Cu2+ by octahedral Ni2+ gives [Ni(WNap)(H2O)2(MeOH)]·H2O, 2, with a slight increase of hydrophilic layer width (by ca. 2 A). The additional hydroxyl group in [Ni(YNap)(H2O)2(MeOH)]·1.5H2O, 3 (YNap = N-(2-hydroxy-1-naphthalidene)-L-tyrosine), successfully introduces a separate amphiphilic layer, originated from the hydrogen bond interaction of tyrosine side chain. The coordination environments of 2 and 3 are isostructural whereas 1 is dinuclear with the Cu(II) metal centers showing different geometries (square planar and square pyramid).

Studying forkhead box protein A1–DNA interaction and ligand inhibition using gold nanoparticles, electrophoretic mobility shift assay, and fluorescence anisotropy

Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. Conventional analytical methods-electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA)-as well as a gold nanoparticles (AuNPs)-based assay were used to study DNA binding properties of FoxA1 and ligand interruption of FoxA1-DNA binding. In the AuNPs assay, the distinct ability of protein-DNA complex to protect AuNPs against salt-induced aggregation was exploited to screen sequence selectivity and determine the binding affinity constant based on AuNPs color change and absorbance spectrum shift. Both conventional EMSA and FA and the AuNPs assay suggested that FoxA1 binds to DNA in a core sequence-dependent manner and the flanking sequence also played a role to influence the affinity. The EMSA and AuNPs were found to be more sensitive than FA in differentiation of sequence-dependent affinity. With the addition of a spin filtration step, AuNPs assay has been extended for studying small molecular ligand inhibition of FoxA1-DNA interactions enabling drug screening. The results correlate very well with those obtained using FA.

Identification of a Wells–Dawson polyoxometalate-based AP-2γ inhibitor with pro-apoptotic activity

AP-2 gamma (AP-2γ) is a transcription factor that plays pivotal roles in breast cancer biology. To search for small molecule inhibitors of AP-2γ, we performed a high-throughput fluorescence anisotropy screen and identified a polyoxometalate compound with Wells-Dawson structure K6[P2Mo18O62] (Dawson-POM) that blocks the DNA-binding activity of AP-2γ. We showed that this blocking activity is due to the direct binding of Dawson-POM to AP-2γ. We also provided evidence to show that Dawson-POM decreases AP-2γ-dependent transcription similar to silencing the gene. Finally, we demonstrated that Dawson-POM contains anti-proliferative and pro-apoptotic effects in breast cancer cells. In summary, we identified the first small molecule inhibitor of AP-2γ and showed Dawson-POM-mediated inhibition of AP-2γ as a potential avenue for cancer therapy.

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 °C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.