Recently, a super uranyl binding protein (SUP) was developed, which exhibits excellent sensitivity/selectivity to bind ions. It can be immobilized onto surface in sensing devices detect Here, sum frequency generation (SFG) vibrational spectroscopy applied probe the interfacial structures of surface-immobilized SUP. The collected SFG spectra were compared calculated orientation-dependent SUP using one-excitonic Hamiltonian approach based on crystal deduce most likely orientation(s)....

Abstract Protein design is a useful strategy to interrogate the protein structure‐function relationship. We demonstrate using highly modular 3‐stranded coiled coil (TRI‐peptide system) that functional type 2 copper center exhibiting nitrite reductase (NiR) activity exhibits highest homogeneous catalytic efficiency under aqueous conditions for reduction of NO and H O. Modification amino acids in second coordination sphere increases up 75‐fold compared previously reported systems. find also...

The equilibrium solubilities of benzoic acid (BA), salicylic (SAL), and acetylsalicylic (ASA) were determined in binary (solute + CO2), ternary (two solutes quaternary systems (three CO2) at temperatures ranging from (308 to 328) K pressures (10.1 28.0) MPa. Solubility data obtained using a dynamic approach with simple reliable apparatus. Polar mixed-solid solute demonstrated solubility enhancements, which consistent the entrainer effect. In all polar studied, least one component exhibited...

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with tightly controlled reduction potential found close range around optimal value 0.36 V versus normal hydrogen electrode (NHE). Rationally designed proteins well-defined three-dimensional structures offer new opportunities obtaining functional SOD mimics....

The reaction of nitric oxide with oxy-myoglobin (oxyMb) to form ferric myoglobin (metMb) and nitrate, the metMb-catalyzed isomerization peroxynitrite have long been assumed proceed via same iron-bound intermediate (metMb(OONO)). More recent research showed that nitrate produces detectable amounts nitrogen dioxide ferryl (ferrylMb). This suggests a mechanism in which binds metMb, ferrylMb is transiently generated by dissociation NO2, formed when NO2 attacks oxo ligand. presence free products...

Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres nuclearity, all cupredoxins share a common Greek Key β-sheet fold. We have previously reported design copper protein within completely distinct three-helical bundle protein, α3DChC2.(1) While this demonstrated β-barrel fold was not requisite...

Copper proteins have the capacity to serve as both redox active catalysts and purely electron transfer centers. A longstanding question in this field is how function of histidine ligated Cu centers are modulated by δ vs ε-nitrogen ligation imidazole. Evaluating impact these coordination modes on structure comparative analysis deposited crystal structures confounded factors such differing protein folds disparate secondary spheres that make direct comparison isomers difficult. Here, we present...

The development of redox-active metalloprotein catalysts is a challenging objective de novo protein design. Within this Perspective we detail our efforts to create Cu nitrite reductase (NiR) by incorporating into the hydrophobic interior well-defined three-stranded coiled coils (3SCCs). scaffold contains three histidine residues that provide layer nitrogen donors mimic type 2 catalytic site NiR. We have found strategy successfully produces an active and stable CuNiR model functions for over...

De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control iron coordination geometry when a heme cofactor is allowed bind either histidine or cysteine ligands, within single artificial protein. Consequently, uncovered reversible pH-induced switch of axial ligation this simplified scaffold. Characterization specific modes was done by using UV/Vis Electron Paramagnetic Resonance...

ConspectusThe relationship between structure and function has long been one of the major points investigation in Biophysics. Understanding how much, or little, a protein's often complicated is necessary for its can lead to directed therapeutic strategies would allow design proteins specific desired functions. Studying protein by de novo builds functionality from ground up completely unrelated noncoded scaffold. Our lab used this strategy study heavy transition metal binding within TRI family...

While many life-critical reactions would be infeasibly slow without metal cofactors, a detailed understanding of how protein structure can influence catalytic activity remains elusive. Using de novo designed three-stranded coiled coils (TRI and Grand peptides formed using heptad repeat approach), we examine the insertion three residue discontinuity, known as stammer insert, directly adjacent to (His)3 binding site alters activity. The stammer, which locally twist helix, significantly...

Blue copper proteins have a constrained Cu(II) geometry that has proven difficult to recapitulate outside native cupredoxin folds. Previous work successfully designed green which could be tuned blue using exogenous ligands, but the question of how one can create self-contained site within de novo scaffold, especially removed from fold, remained. We recently reported red protein three helical bundle scaffold we later revisited and determined nitrosocyanin mimic, with CuHis2CysGlu binding...

Bilin-binding fluorescent proteins like UnaG–bilirubin are noncovalent ligand-dependent reporters for oxygen-free microscopy but restricted to blue and far-red fluorescence. Here we describe a high-throughput screening approach provide new UnaG–ligand pair that can be excited in the 532 nm green excitation channel. We identified novel orange maximally emits at 581 nm. Whereas benzothiazole-based ligand itself is nominally fluorescent, compound binds UnaG with high affinity (Kd = 3 nM) induce...

The previously reported nitric oxide precursor [Mn(PaPy2Q)NO]ClO4 (1), where (PaPy2QH) is N,N-bis(2-pyridylmethyl)-amine-N-ethyl-2-quinoline-2-carboxamide, was used to investigate the interaction between NO and protein truncated hemoglobin N (trHbN) from pathogen Mycobacterium tuberculosis. Oxy-trHbN exceptionally efficient at converting nitrate, with a rate constant of 7.45 × 108 M–1 s–1 [Ouellet, H., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5902] compared 4 107 for oxy-myoglobin...

Abstract Protein design is a useful strategy to interrogate the protein structure‐function relationship. We demonstrate using highly modular 3‐stranded coiled coil (TRI‐peptide system) that functional type 2 copper center exhibiting nitrite reductase (NiR) activity exhibits highest homogeneous catalytic efficiency under aqueous conditions for reduction of NO and H O. Modification amino acids in second coordination sphere increases up 75‐fold compared previously reported systems. find also...

Ferulic acid decarboxylase catalyzes the decarboxylation of various substituted phenylacrylic acids to their corresponding styrene derivatives and CO2 using recently discovered cofactor prenylated FMN (prFMN). The mechanism involves an unusual 1,3-dipolar cycloaddition reaction between prFMN substrate generate a cycloadduct capable undergoing decarboxylation. Using native mass spectrometry, we show enzyme forms stable prFMN–styrene that accumulates on during turnover. Pre-steady state...

The design of metal-binding sites in proteins that combine high affinity with selectivity for the desired metal ion remains a challenging goal. Recently, protein designed to display femtomolar UO22+, dubbed "Super Uranyl-binding Protein" (SUP), was described, potential applications removing UO22+ water. Although it discriminated most ions present seawater, showed surprisingly Cu2+ ions. Here, we have investigated binding SUP using combination electron paramagnetic resonance, fluorescence and...

Abstract Viperin is a radical S-adenosylmethionine enzyme that catalyzes the formation of antiviral ribonucleotide, 3’-deoxy-3’,4’-didehydroCTP. The conserved across all kingdoms life, and in higher animals viperin localized to ER-membrane lipid droplets through an N-terminal extension forms amphipathic helix. Evidence suggests plays important role viperin’s interactions with other membrane proteins. These serve modulate activity various enzymes are for viral replication constitute another...