Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer.

Technologically critical rare-earth elements are notoriously difficult to separate, owing to their subtle differences in ionic radius and coordination number1-3. The natural lanthanide-binding protein lanmodulin (LanM)4,5 is a sustainable alternative to conventional solvent-extraction-based separation6. Here we characterize a new LanM, from Hansschlegelia quercus (Hans-LanM), with an oligomeric state sensitive to rare-earth ionic radius, the lanthanum(III)-induced dimer being >100-fold tighter than the dysprosium(III)-induced dimer. X-ray crystal structures illustrate how picometre-scale differences in radius between lanthanum(III) and dysprosium(III) are propagated to Hans-LanM's quaternary structure through a carboxylate shift that rearranges a second-sphere hydrogen-bonding network. Comparison to the prototypal LanM from Methylorubrum extorquens reveals distinct metal coordination strategies, rationalizing Hans-LanM's greater selectivity within the rare-earth elements. Finally, structure-guided mutagenesis of a key residue at the Hans-LanM dimer interface modulates dimerization in solution and enables single-stage, column-based separation of a neodymium(III)/dysprosium(III) mixture to >98% individual element purities. This work showcases the natural diversity of selective lanthanide recognition motifs, and it reveals rare-earth-sensitive dimerization as a biological principle by which to tune the performance of biomolecule-based separation processes.

An Unusual Ferryl Intermediate and Its Implications for the Mechanism of Oxacyclization by the Loline-Producing Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, LolO.

N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H· abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H· from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.

Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a ∼4-ps and a ∼20-ps population to produce the charge-separated state P+HA- in all variants. Global analysis indicates that in the ∼4-ps population, P+HA- forms through a two-step process, P*→ P+BA-→ P+HA-, while in the ∼20-ps population, it forms via a one-step P* → P+HA- superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from ∼25 to ∼45% among variants, while in WT, this percentage is ∼15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P+BA- intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an ∼110-meV increase in the free energy of P+BA- along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P+BA- formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.

Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States.

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H•) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationic l-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l-Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme.

The past decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or nonbiological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most redesigned proteins. While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional ground-state catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an "excited-state enzyme". Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs. As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics tailored for specific applications could be predicted and then demonstrated. The underlying energetic landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.

Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore.

The neutral or A state of the green fluorescent protein (GFP) chromophore is a remarkable example of a photoacid naturally embedded in the protein environment and accounts for the large Stokes shift of GFP in response to near UV excitation. Its color tuning mechanism has been largely overlooked, as it is less preferred for imaging applications than the redder anionic or B state. Past studies, based on site-directed mutagenesis or solvatochromism of the isolated chromophore, have concluded that its color tuning range is much narrower than its anionic counterpart. However, as we performed extensive investigation on more GFP mutants, we found that the color of the neutral chromophore can be more sensitive to protein electrostatics than can the anionic counterpart. Electronic Stark spectroscopy reveals a fundamentally different electrostatic color tuning mechanism for the neutral state of the chromophore that demands a three-form model as compared to that of the anionic state, which requires only two forms ( J. Am. Chem. Soc. 2019, 141, 15250-15265). Specifically, an underlying zwitterionic charge-transfer state is required to explain its sensitivity to electrostatics. As the Stokes shift is tightly linked to excited-state proton transfer (ESPT) of the protonated chromophore, we infer design principles of the GFP chromophore as a photoacid through the color tuning mechanisms of both protonation states. The three-form model could also be applied to similar biological and nonbiological dyes and complements the failure of the two-form model for donor-acceptor systems with localized ground-state electronic distributions.

Structural and spectroscopic characterization of photoactive yellow protein and photoswitchable fluorescent protein constructs containing heavy atoms.

Photo-induced structural rearrangements of chromophore-containing proteins are essential for various light-dependent signaling pathways and optogenetic applications. Ultrafast structural and spectroscopic methods have offered insights into these structural rearrangements across many timescales. However, questions still remain about exact mechanistic details, especially regarding photoisomerization of the chromophore within these proteins femtoseconds to picoseconds after photoexcitation. Instrumentation advancements for time-resolved crystallography and ultrafast electron diffraction provide a promising opportunity to study these reactions, but achieving enough signal-to-noise is a constant challenge. Here we present four new photoactive yellow protein constructs and one new fluorescent protein construct that contain heavy atoms either within or around the chromophore and can be expressed with high yields. Structural characterization of these constructs, most at atomic resolution, show minimal perturbation caused by the heavy atoms compared to wild-type structures. Spectroscopic studies report the effects of the heavy atom identity and location on the chromophore's photophysical properties. None of the substitutions prevent photoisomerization, although certain rates within the photocycle may be affected. Overall, these new proteins containing heavy atoms are ideal samples for state-of-theart time-resolved crystallography and electron diffraction experiments to elucidate crucial mechanistic information of photoisomerization.

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs).

Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached ß-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.

Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins.

Green fluorescent proteins (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting that all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all of the observed strong correlations among photophysical properties; related subtopics are extensively discussed in the Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating the photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue that this model should also be generally applicable to both biological and nonbiological polymethine dyes.

Synergistic Binding of the Halide and Cationic Prime Substrate of the l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States.

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl - or Br - ), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three non-gaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O 2 . Halide, 2OG, and (lastly) O 2 all coordinate directly to the cofactor to initiate its conversion to a cis -halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H•) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l -lysine 4-chlorinase, BesD. After 2OG adds, subsequent coordination of the halide to the cofactor and binding of cationic l -Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon addition of O 2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l -Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate• l -Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l -Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply that (i) BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l -Lys binding and chloride coordination following loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

Optical control of ultrafast structural dynamics in a fluorescent protein.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump-probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.

Use of Noncanonical Tyrosine Analogues to Probe Control of Radical Intermediates during Endoperoxide Installation by Verruculogen Synthase (FtmOx1).

Important bioactive natural products, including prostaglandin H2 and artemisinin, contain reactive endoperoxides. Known enzymatic pathways for endoperoxide installation require multiple hydrogen-atom transfers (HATs). For example, iron(II)- and 2-oxoglutarate-dependent verruculogen synthase (FtmOx1; EC 1.14.11.38) mediates HAT from aliphatic C21 of fumitremorgin B, capture of O2 by the C21 radical (C21•), addition of the peroxyl radical (C21-O-O•) to olefinic C27, and HAT to the resultant C26•. Recent studies proposed conflicting roles for FtmOx1 tyrosine residues, Tyr224 and Tyr68, in the HATs from C21 and to C26•. Here, analysis of variant proteins bearing a ring-halogenated tyrosine or (amino)phenylalanine in place of either residue establishes that Tyr68 is the hydrogen donor to C26•, while Tyr224 has no essential role. The radicals that accumulate rapidly in FtmOx1 variants bearing a HAT-competent tyrosine analog at position 68 exhibit hypsochromically shifted absorption and, in cases of fluorine substitution, 19F-coupled electron-paramagnetic-resonance (EPR) spectra. By contrast, functional Tyr224-substituted variants generate radicals with unaltered light-absorption and EPR signatures as they produce verruculogen. The alternative major product of the Tyr68Phe variant, which forms competitively with verruculogen also in wild-type FtmOx1 in 2H2O and in the variant with the less readily oxidized 2,3-F2Tyr at position 68, is identified by mass spectrometry and isotopic labeling as the 26-hydroxy-21,27-endoperoxide compound formed after capture of another equivalent of O2 by the longer lived C26•. The results highlight the considerable chemical challenges the enzyme must navigate in averting both oxygen rebound and a second O2 coupling to obtain verruculogen selectively over other possible products.

Unusual Spectroscopic and Electric Field Sensitivity of Chromophores with Short Hydrogen Bonds: GFP and PYP as Model Systems.

Short hydrogen bonds, with heavy-atom distances less than 2.7 Å, are believed to exhibit proton delocalization, and their possible role in catalysis has been widely debated. While spectroscopic and/or structural methods are usually employed to study the degree of proton delocalization, ambiguities still arise, and no direct information on the corresponding potential energy surface is obtained. Here, we apply an external electric field to perturb the short hydrogen bond(s) within a collection of green fluorescent protein S65T/H148D variants and photoactive yellow protein mutants, where the chromophore participates in the short hydrogen bond(s) and serves as an optical probe of the proton position. As the proton is charged, its position may shift in response to the external electric field, and the chromophore's electronic absorption can thus reflect the ease of proton transfer. The results suggest that low-barrier hydrogen bonds (LBHBs) are not present within these proteins even when proton affinities between donor and acceptor are closely matched. Exploiting the chromophores as precalibrated electrostatic probes, the covalency of short hydrogen bonds as a nonelectrostatic component is also revealed. A theoretical framework is developed to address a possible contribution of unusually large polarizabilities of short hydrogen bonds due to proton delocalization, but no clear evidence for this phenomenon is found in accordance with the absence of LBHBs.

Electrostatic control of photoisomerization pathways in proteins.

Rotation around a specific bond after photoexcitation is central to vision and emerging opportunities in optogenetics, super-resolution microscopy, and photoactive molecular devices. Competing roles for steric and electrostatic effects that govern bond-specific photoisomerization have been widely discussed, the latter originating from chromophore charge transfer upon excitation. We systematically altered the electrostatic properties of the green fluorescent protein chromophore in a photoswitchable variant, Dronpa2, using amber suppression to introduce electron-donating and electron-withdrawing groups to the phenolate ring. Through analysis of the absorption (color), fluorescence quantum yield, and energy barriers to ground- and excited-state isomerization, we evaluate the contributions of sterics and electrostatics quantitatively and demonstrate how electrostatic effects bias the pathway of chromophore photoisomerization, leading to a generalized framework to guide protein design.

Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor.

Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3'-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.

Planting hope in loss and grief: self-care applications of horticultural therapy for grief caregivers in Taiwan.

In 2008, the Taiwan Association for Care and Counseling for Loss organized a workshop about Horticultural Therapy, conducted as a participatory action research (PAR). Nineteen grief caregivers participated. Specific goals were designed according to a survey of participant expectations and focus-group discussions. The workshop content included lectures and interactive activities. Results demonstrated that most participants displayed an increased awareness of personal loss and meaning in grief, indicating that horticulture and nature appreciation might relieve individual grief and stress. The report introduces the rationale, evolution, execution, and results of the program development.

The experience of perioperative nurses involved in organ procurement.

BACKGROUND: In the organ transplant process, the perioperative nurse plays an important role in assisting with organ procurement, during which they are frequently required to witness the death of donors. Their experiences and feelings regarding such are largely hidden and little discussed. PURPOSE: The purpose of this study was to understand the experience, feelings, and self-care strategies of perioperative nurses during the course of organ procurement. METHODS: This study adopted a qualitative method, using purposeful sampling methods and a semiconstructed outline to conduct face-to-face interviews with six perioperative nurses from an organ procurement organization located in northern Taiwan. Collected data were analyzed using content analysis. RESULTS: Results were categorized into two distinct parts. The first described the organ procurement experience, with described feelings including a journey begins with learning, feelings of slaughter and doubts about death, and death is a new beginning. The second described ideas of self-care, with described ideas including facing problems, thinking and adjusting, engaging in leisure activities, holding religious beliefs, separating work from private time, continuing self-training, and sharing. CONCLUSIONS: This study indicates that witnessing donor deaths makes perioperative nurses feel uncomfortable and even induces trauma. Better understanding the effects of the organ donation process on all related staff and how to facilitate their self-care will be important parts of the next step in helping perioperative nurses better cope with their work environment and become better equipped to balance professional and psychological needs.