Photoacid Dynamics in the Green Fluorescent Protein.

The photoacid dynamics of fluorescent proteins include both electronic excited- and ground-state mechanisms of proton transfer. The associated characteristic timescales of these reactions range over many orders of magnitude, and the tunneling, barrier crossing, and relevant thermodynamics have in certain cases been linked to coherent nuclear motion. We review the literature and summarize the experiments and theory that demonstrate proton tunneling in the electronic ground state of the green fluorescent protein (GFP). We also discuss the excited-state proton-transfer reaction of GFP that takes place on the picosecond timescale. Although this reaction has been investigated using several vibrational spectroscopic methods, the interpretation remains unsettled. We discuss recent advances as well as remaining questions, in particular those related to the vibrational mode couplings that involve low-frequency modulations of chromophore vibrations on the timescale of proton transfer.

Electrical unfolding of cytochrome c during translocation through a nanopore constriction.

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.

Environmental filtering of native and non-native stream macrophyte assemblages by habitat disturbances in an agricultural landscape.

Understanding how inter-specific variation in functional traits affects native and non-native species responses to stream disturbances, is necessary to inform management strategies, providing tools for biomonitoring, conservation and restoration. This study used a functional trait approach to characterise the responses of macrophyte assemblages to reach-scale disturbances (measured by lack of riparian shading, altered hydromorphology and eutrophication), from 97 wadeable stream sites in an agriculturally impacted region of New Zealand. To determine whether macrophyte assemblages differed due to disturbances, we examined multidimensional assemblage functional structure in relation to eleven functional traits and further related two functional diversity indices (entropy and originality) to disturbances. Macrophyte assemblages showed distinct patterns in response to disturbances, with riparian shading and hydromorphological conditions being the strongest variables shaping macrophyte functional structure. In the multidimensional space, most of the non-native species were associated with disturbed conditions. These species had traits allowing faster colonisation rates (higher number of reproductive organs and larger root-rhizome system) and superior competitive abilities for resources (tall and dense canopy, heterophylly and greater preferences for light and nitrogen). In addition, lack of riparian shading increased the abundance of functionally distinct species (i.e. entropy), and eutrophication resulted in the growth of functionally unique species (i.e. originality). We demonstrated that stream reach-scale habitat disturbances were associated to a dominance of more productive species, equating to a greater abundance of non-native species. This, can result in a displacement of native species, habitat alterations, and changes to higher trophic level assemblages. Our results suggests that reach-scale management efforts such as the conservation and restoration of riparian vegetation that provides substantial shading and hydromorphologically diverse in-stream habitat, would have beneficial direct and indirect effects on ecosystem functioning, and contribute to the mitigation of land-use impacts.

Oxygen-Induced In Situ Manipulation of the Interlayer Coupling and Exciton Recombination in Bi2Se3/MoS2 2D Heterostructures.

Two-dimensional (2D) heterostructures are more than a sum of the parent 2D materials, but are also a product of the interlayer coupling, which can induce new properties. In this paper, we present a method to tune the interlayer coupling in Bi2Se3/MoS2 2D heterostructures by regulating the oxygen presence in the atmosphere, while applying laser or thermal energy. Our data suggest that the interlayer coupling is tuned through the diffusive intercalation and deintercalation of oxygen molecules. When one layer of Bi2Se3 is grown on monolayer MoS2, an influential interlayer coupling is formed, which quenches the signature photoluminescence (PL) peaks. However, thermally treating in the presence of oxygen disrupts the interlayer coupling, facilitating the emergence of the MoS2 PL peak. Our density functional theory calculations predict that intercalated oxygen increases the interlayer separation ∼17%, disrupting the interlayer coupling and inducing the layers to behave more electronically independent. The interlayer coupling can then be restored by thermally treating in N2 or Ar, where the peaks will requench. Hence, this is an interesting oxygen-induced switching between "non-radiative" and "radiative" exciton recombination. This switching can also be accomplished locally, controllably, and reversibly using a low-power focused laser, while changing the environment from pure N2 to air. This allows for the interlayer coupling to be precisely manipulated with submicron spatial resolution, facilitating site-programmable 2D light-emitting pixels whose emission intensity could be precisely varied by a factor exceeding 200×. Our results show that these atomically thin 2D heterostructures may be excellent candidates for oxygen sensing.

Adiabatic Ligand Binding in Heme Proteins: Ultrafast Kinetics of Methionine Rebinding in Ferrous Cytochrome c.

The dynamics of methionine geminate recombination following photodissociation in ferrous cytochrome c is investigated over a broad temperature range. The kinetic response, above the solvent glass transition ( Tg), is nearly monoexponential and displays a weak temperature dependence. Below Tg, the rebinding kinetics are nonexponential and can be explained using a quenched distribution of enthalpic rebinding barriers, arising from a relatively narrow distribution of heme out-of-plane displacements. The Arrhenius prefactor of this (Δ S = 2) reaction is ∼1011 s-1, which is similar to what has been found for the (Δ S = 1) NO binding reaction in heme proteins. This observation, along with other examples of ultrafast CO binding, provides strong evidence that ligand binding to heme is an adiabatic reaction with a spin-independent prefactor. In order to simultaneously account for the adiabatic nature of the reaction as well as the temperature dependence of both ultrafast CO and methionine geminate rebinding, it is proposed that a spin triplet state intersects and strongly couples to the reactant ( S = 2) and product ( S = 0) state surfaces in the transition state region along the reaction coordinate. It is also suggested that the nature of the intersecting triplet state and the reaction path may depend upon the proximity of the photolyzed ligand relative to the iron atom. At temperatures below ∼60 K, the kinetic data suggest that there is either an unexpected retardation of the heme photoproduct relaxation or that heavy atom quantum mechanical tunneling becomes significant.

Ultrafast CO Kinetics in Heme Proteins: Adiabatic Ligand Binding and Heavy Atom Tunneling.

The ultrafast kinetics of CO rebinding to carbon monoxide oxidation activator protein (ChCooA) are measured over a wide temperature range and compared with the kinetics of CO binding in other heme systems such as myoglobin (Mb) and hemoglobin (Hb). The Arrhenius prefactor for CO binding to ChCooA and protoheme (∼1011 s-1) is similar to what is found for spin-allowed NO binding to heme proteins and is several orders of magnitude larger than the prefactor of Mb and Hb (∼109 s-1). This indicates that the CO binding reaction is adiabatic, in contrast to the commonly held view that it is nonadiabatic due to spin-forbidden (ΔS = 2) selection rules. Under the adiabatic condition, entropic factors, rather than spin-selection rules, are the source of the reduced Arrhenius prefactors associated with CO binding in Mb and Hb. The kinetic response of ChCooA-CO is nonexponential at all temperatures, including 298 K, and is described quantitatively using a distribution of enthalpic rebinding barriers associated with heterogeneity in the heme doming conformation. Above the solvent glass transition (Tg ∼ 180 K), the rebinding progress slows as temperature increases, and this is ascribed to an evolution of the distribution toward increased heme doming and larger enthalpic barriers. Between Tg and ∼60 K, the nonexponential rebinding slows down as the temperature is lowered and the survival fraction follows the predictions expected for a quenched barrier distribution. Below ∼60 K the rebinding kinetics do not follow these predictions unless quantum mechanical tunneling along the heme doming coordinate is also included as an active channel for CO binding.

Tunneling Kinetics and Nonadiabatic Proton-Coupled Electron Transfer in Proteins: The Effect of Electric Fields and Anharmonic Donor-Acceptor Interactions.

A proper description of proton donor-acceptor (D-A) distance fluctuations is crucial for understanding tunneling in proton-coupled electron transport (PCET). The typical harmonic approximation for the D-A potential results in a Gaussian probability distribution, which does not appropriately reflect the electronic repulsion forces that increase the energetic cost of sampling shorter D-A distances. Because these shorter distances are the primary channel for thermally activated tunneling, the analysis of tunneling kinetics depends sensitively on the inherently anharmonic nature of the D-A interaction. Thus, we have used quantum chemical calculations to account for the D-A interaction and developed an improved model for the analysis of experimental tunneling kinetics. Strong internal electric fields are also considered and found to contribute significantly to the compressive forces when the D-A distance distribution is positioned below the van der Waals contact distance. This model is applied to recent experiments on the wild type (WT) and a double mutant (DM) of soybean lipoxygenase-1 (SLO). The compressive force necessary to prepare the tunneling-active distribution in WT SLO is found to fall in the ∼ nN range, which greatly exceeds the measured values of molecular motor and protein unfolding forces. This indicates that ∼60-100 MV/cm electric fields, aligned along the D-A bond axis, must be generated by an enzyme conformational interconversion that facilitates the PCET tunneling reaction. Based on the absolute value of the measured tunneling rate, and using previously calculated values of the electronic matrix element, the population of this tunneling-active conformation is found to lie in the range 10-5-10-7, indicating this is a rare structural fluctuation that falls well below the detection threshold of recent ENDOR experiments. Additional analysis of the DM tunneling kinetics leads to a proposal that a disordered (high entropy) conformation could be tunneling-active due to its broad range of sampled D-A distances.

Peptide-Decorated Tunable-Fluorescence Graphene Quantum Dots.

We report here the synthesis of graphene quantum dots with tunable size, surface chemistry, and fluorescence properties. In the size regime 15-35 nm, these quantum dots maintain strong visible light fluorescence (mean quantum yield of 0.64) and a high two-photon absorption (TPA) cross section (6500 Göppert-Mayer units). Furthermore, through noncovalent tailoring of the chemistry of these quantum dots, we obtain water-stable quantum dots. For example, quantum dots with lysine groups bind strongly to DNA in solution and inhibit polymerase-based DNA strand synthesis. Finally, by virtue of their mesoscopic size, the quantum dots exhibit good cell permeability into living epithelial cells, but they do not enter the cell nucleus.

Proton-Coupled Electron Transfer and the "Linear Approximation" for Coupling to the Donor-Acceptor Distance Fluctuations.

The often-used "linear approximation" for treating the coupling of proton donor-acceptor (D-A) distance fluctuations to proton-coupled electron transfer tunneling reactions is systematically examined. The accuracy of this approximation is found to depend on the potential energy surfaces that are used to describe both the tunneling particle vibrations and the D-A coordinate probability distribution. Harmonic treatment of both the tunneling particle and the D-A coordinates results in a significant breakdown of the linear approximation when the width of the D-A distribution exceeds ∼0.05 Å. When a symmetric back-to-back Morse potential is used to describe the tunneling particle vibrations in the reactant and product states, the D-A distribution width can be expanded up to ∼0.09 Å before the rates calculated using the linear approximation exceed the exact result by an order of magnitude. Incorporation of a more realistic anharmonic D-A potential, based on quantum calculations, includes the important electronic D-A repulsion energy so that the sampling of short D-A distances is reduced. This approach improves the linear approximation as long as the D-A distribution has a width less than ∼0.1 Å. The conditions for the validity of the linear approximation are found to be more fragile when the calculated kinetic isotope effect (KIE) and its temperature dependence are also taken into account.

Wide-dynamic-range kinetic investigations of deep proton tunnelling in proteins.

Directional proton transport along 'wires' that feed biochemical reactions in proteins is poorly understood. Amino-acid residues with high pKa are seldom considered as active transport elements in such wires because of their large classical barrier for proton dissociation. Here, we use the light-triggered proton wire of the green fluorescent protein to study its ground-electronic-state proton-transport kinetics, revealing a large temperature-dependent kinetic isotope effect. We show that 'deep' proton tunnelling between hydrogen-bonded oxygen atoms with a typical donor-acceptor distance of 2.7-2.8 Šfully accounts for the rates at all temperatures, including the unexpectedly large value (2.5 × 10(9) s(-1)) found at room temperature. The rate-limiting step in green fluorescent protein is assigned to tunnelling of the ionization-resistant serine hydroxyl proton. This suggests how high-pKa residues within a proton wire can act as a 'tunnel diode' to kinetically trap protons and control the direction of proton flow.

Kinetic Control of O2 Reactivity in H-NOX Domains.

Transient absorption, resonance Raman, and vibrational coherence spectroscopies are used to investigate the mechanisms of NO and O2 binding to WT Tt H-NOX and its P115A mutant. Vibrational coherence spectra of the oxy complexes provide clear evidence for the enhancement of an iron-histidine mode near 217 cm(-1) following photoexcitation, which indicates that O2 can be dissociated in these proteins. However, the quantum yield of O2 photolysis is low, particularly in the wild type (≲3%). Geminate recombination of O2 and NO in both of these proteins is very fast (∼1.4 × 10(11) s(-1)) and highly efficient. We show that the distal heme pocket of the H-NOX system forms an efficient trap that limits the O2 off-rate and determines the overall affinity. The distal pocket hydrogen bond, which appears to be stronger in the P115A mutant, may help retard the O2 ligand from escaping into the solvent following either photoinduced or thermal dissociation. This, along with a strengthening of the Fe-O2 bond that is correlated with the significant heme ruffing and saddling distortions, explains the unusually high O2 affinity of WT Tt H-NOX and the even higher affinity found in the P115A mutant.

Deep proton tunneling in the electronically adiabatic and non-adiabatic limits: comparison of the quantum and classical treatment of donor-acceptor motion in a protein environment.

Analytical models describing the temperature dependence of the deep tunneling rate, useful for proton, hydrogen, or hydride transfer in proteins, are developed and compared. Electronically adiabatic and non-adiabatic expressions are presented where the donor-acceptor (D-A) motion is treated either as a quantized vibration or as a classical "gating" distribution. We stress the importance of fitting experimental data on an absolute scale in the electronically adiabatic limit, which normally applies to these reactions, and find that vibrationally enhanced deep tunneling takes place on sub-ns timescales at room temperature for typical H-bonding distances. As noted previously, a small room temperature kinetic isotope effect (KIE) does not eliminate deep tunneling as a major transport channel. The quantum approach focuses on the vibrational sub-space composed of the D-A and hydrogen atom motions, where hydrogen bonding and protein restoring forces quantize the D-A vibration. A Duschinsky rotation is mandated between the normal modes of the reactant and product states and the rotation angle depends on the tunneling particle mass. This tunnel-mass dependent rotation contributes substantially to the KIE and its temperature dependence. The effect of the Duschinsky rotation is solved exactly to find the rate in the electronically non-adiabatic limit and compared to the Born-Oppenheimer (B-O) approximation approach. The B-O approximation is employed to find the rate in the electronically adiabatic limit, where we explore both harmonic and quartic double-well potentials for the hydrogen atom bound states. Both the electronically adiabatic and non-adiabatic rates are found to diverge at high temperature unless the proton coupling includes the often neglected quadratic term in the D-A displacement from equilibrium. A new expression is presented for the electronically adiabatic tunnel rate in the classical limit for D-A motion that should be useful to experimentalists working near room temperature. This expression also holds when a broad protein conformational distribution of D-A equilibrium distances dominates the spread of the D-A vibrational wavefunction.

Investigations of the low frequency modes of ferric cytochrome c using vibrational coherence spectroscopy.

Femtosecond vibrational coherence spectroscopy is used to investigate the low frequency vibrational dynamics of the electron transfer heme protein, cytochrome c (cyt c). The vibrational coherence spectra of ferric cyt c have been measured as a function of excitation wavelength within the Soret band. Vibrational coherence spectra obtained with excitation between 412 and 421 nm display a strong mode at ~44 cm(-1) that has been assigned to have a significant contribution from heme ruffling motion in the electronic ground state. This assignment is based partially on the presence of a large heme ruffling distortion in the normal coordinate structural decomposition (NSD) analysis of the X-ray crystal structures. When the excitation wavelength is moved into the ~421-435 nm region, the transient absorption increases along with the relative intensity of two modes near ~55 and 30 cm(-1). The intensity of the mode near 44 cm(-1) appears to minimize in this region and then recover (but with an opposite phase compared to the blue excitation) when the laser is tuned to 443 nm. These observations are consistent with the superposition of both ground and excited state coherence in the 421-435 nm region due to the excitation of a weak porphyrin-to-iron charge transfer (CT) state, which has a lifetime long enough to observe vibrational coherence. The mode near 55 cm(-1) is suggested to arise from ruffling in a transient CT state that has a less ruffled heme due to its iron d(6) configuration.

Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c.

Cytochrome (cyt) c is an important electron transfer protein. The ruffling deformation of its heme cofactor has been suggested to relate to its electron transfer rate. However, there is no direct experimental evidence demonstrating this correlation. In this work, we studied Pseudomonas aeruginosa cytochrome c551 and its F7A mutant. These two proteins, although similar in their X-ray crystal structure, display a significant difference in their heme out-of-plane deformations, mainly along the ruffling coordinate. Resonance Raman and vibrational coherence measurements also indicate significant differences in ruffling-sensitive modes, particularly the low-frequency γa mode found between ∼50-60 cm(-1). This supports previous assignments of γa as having a large ruffling content. Measurement of the photoreduction kinetics finds an order of magnitude decrease of the photoreduction cross-section in the F7A mutant, which has nearly twice the ruffling deformation as the WT. Additional measurements on cytochrome c demonstrate that heme ruffling is correlated exponentially with the electron transfer rates and suggest that ruffling could play an important role in redox control. A major relaxation of heme ruffling in cytochrome c, upon binding to the mitochondrial membrane, is discussed in this context.

Analytical Harmonic Vibrational Frequencies for the Green Fluorescent Protein Computed with ONIOM: Chromophore Mode Character and Its Response to Environment.

A systematic comparison of different environmental effects on the vibrational modes of the 4-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) chromophore using the ONIOM method allows us to model how the molecule's spectroscopic transitions are modified in the Green Fluorescent Protein (GFP). ONIOM(QM:MM) reduces the expense of normal mode calculations when computing the majority of second derivatives only at the MM level. New developments described here for the efficient solution of the CPHF equations, including contributions from electrostatic interactions with environment charges, mean that QM model systems of ∼100 atoms can be embedded within a much larger MM environment of ∼5000 atoms. The resulting vibrational normal modes, their associated frequencies, and dipole derivative vectors have been used to interpret experimental difference spectra (GFPI2-GFPA), chromophore vibrational Stark shifts, and changes in the difference between electronic and vibrational transition dipoles (mode angles) in the protein environment.

Investigations of heme ligation and ligand switching in cytochromes p450 and p420.

It is generally accepted that the inactive P420 form of cytochrome P450 (CYP) involves the protonation of the native cysteine thiolate to form a neutral thiol heme ligand. On the other hand, it has also been suggested that recruitment of a histidine to replace the native cysteine thiolate ligand might underlie the P450 → P420 transition. Here, we discuss resonance Raman investigations of the H93G myoglobin (Mb) mutant in the presence of tetrahydrothiophene (THT) or cyclopentathiol (CPSH), and on pressure-induced cytochrome P420cam (CYP101), that show a histidine becomes the heme ligand upon CO binding. The Raman mode near 220 cm⁻¹, normally associated with the Fe-histidine vibration in heme proteins, is not observed in either reduced P420cam or the reduced H93G Mb samples, indicating that histidine is not the ligand in the reduced state. The absence of a mode near 220 cm⁻¹ is also inconsistent with a generalization of the suggestion that the 221 cm⁻¹ Raman mode, observed in the P420-CO photoproduct of inducible nitric oxide synthase (iNOS), arises from a thiol-bound ferrous heme. This leads us to assign the 218 cm⁻¹ mode observed in the 10 ns P420cam-CO photoproduct Raman spectrum to a Fe-histidine vibration, in analogy to many other histidine-bound heme systems. Additionally, the inverse correlation plots of the νFe-His and νCO frequencies for the CO adducts of P420cam and the H93G analogs provide supporting evidence that histidine is the heme ligand in the P420-CO-bound state. We conclude that, when CO binds to the ferrous P420 state, a histidine ligand is recruited as the heme ligand. The common existence of an HXC-Fe motif in many CYP systems allows the C → H ligand switch to occur with only minor conformational changes. One suggested conformation of P420-CO involves the addition of another turn in the proximal L helix so that, when the protonated Cys ligand is dissociated from the heme, it can become part of the helix, and the heme is ligated by the His residue from the adjoining loop region. In other systems, such as iNOS and CYP3A4 (where the HXC-Fe motif is not found), a somewhat larger conformational change would be necessary to recuit a nearby histidine.

Investigations of the low-frequency spectral density of cytochrome c upon equilibrium unfolding.

The equilibrium unfolding process of ferric horse heart cytochrome c (cyt c), induced by guanidinium hydrochloride (GdHCl), was studied using UV-vis absorption spectroscopy, resonance Raman spectroscopy, and vibrational coherence spectroscopy (VCS). The unfolding process was successfully fit using a three-state model which included the fully folded (N) and unfolded (U) states, along with an intermediate (I) assigned to a Lys bound heme. The VCS spectra revealed for the first time several low-frequency heme modes that are sensitive to cyt c unfolding: γ(a) (~50 cm(-1)), γ(b) (~80 cm(-1)), γ(c) (~100 cm(-1)), and ν(s)(His-Fe-His) at 205 cm(-1). These out-of-plane modes have potential functional relevance and are activated by protein-induced heme distortions. The free energies for the N-I and the I-U transitions at pH 7.0 and 20 °C were found to be 4.6 kcal/M and 11.6 kcal/M, respectively. Imidazole was also introduced to replace the methionine ligand so the unfolding can be modeled as a two-state system. The intensity of the mode γ(b)~80 cm(-1) remains nearly constant during the unfolding process, while the amplitudes of the other low frequency modes track with spectral changes observed at higher frequency. This confirms that the heme deformation changes are coupled to the protein tertiary structural changes that take place upon unfolding. These studies also reveal that damping of the coherent oscillations depends sensitively on the coupling between heme and the surrounding water solvent.

Investigations of ferric heme cyanide photodissociation in myoglobin and horseradish peroxidase.

The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has definitively been observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881-1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252-2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75%, and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate of kBA ≈ (3.6 ps)(-1) is the dominant process, some CN(-) exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN(-) association rate, we find that the CN(-) escape rate from the ferric myoglobin pocket to the solution at 293 K is kout ≈ (1-2) × 10(7) s(-1). This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1 × 10(7) s(-1)) under the same conditions. The analysis leads to an escape probability kout/(kout + kBA) ~ 10(-4), which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN(-) bimolecular association rate (170 M(-1) s(-1)), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN(-) bimolecular association rate is larger by ~10(3), making the CN(-) photolysis more difficult to observe.

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa.

The reversible photoswitching between the 'on' and 'off' states of the fluorescent protein Dronpa involves photoisomerization as well as protein side-chain rearrangements, but the process of interconversion remains poorly characterized. Here we use time-resolved infrared measurements to monitor the sequence of these structural changes, but also of proton transfer events, which are crucial to the development of spectroscopic contrast. Light-induced deprotonation of the chromophore phenolic oxygen in the off state is a thermal ground-state process, which follows ultrafast (9 ps) trans-cis photoisomerization, and so does not involve excited-state proton transfer. Steady-state infrared difference measurements exclude protonation of the imidazolinone nitrogen in both the on and off states. Pump-probe infrared measurements of the on state reveal a weakening of the hydrogen bonding between Arg66 and the chromophore C=O, which could be central to initiating structural rearrangement of Arg66 and His193 coinciding with the low quantum yield cis-trans photoisomerization.

Weed risk assessment for aquatic plants: modification of a New Zealand system for the United States.

We tested the accuracy of an invasive aquatic plant risk assessment system in the United States that we modified from a system originally developed by New Zealand's Biosecurity Program. The US system is comprised of 38 questions that address biological, historical, and environmental tolerance traits. Values associated with each response are summed to produce a total score for each species that indicates its risk of invasion. To calibrate and test this risk assessment, we identified 39 aquatic plant species that are major invaders in the continental US, 31 species that have naturalized but have no documented impacts (minor invaders), and 60 that have been introduced but have not established. These species represent 55 families and span all aquatic plant growth forms. We found sufficient information to assess all but three of these species. When the results are compared to the known invasiveness of the species, major invaders are distinguished from minor and non-invaders with 91% accuracy. Using this approach, the US aquatic weed risk assessment correctly identifies major invaders 85%, and non-invaders 98%, of the time. Model validation using an additional 10 non-invaders and 10 invaders resulted in 100% accuracy for the former, and 80% accuracy for the latter group. Accuracy was further improved to an average of 91% for all groups when the 17% of species with scores of 31-39 required further evaluation prior to risk classification. The high accuracy with which we can distinguish non-invaders from harmful invaders suggests that this tool provides a feasible, pro-active system for pre-import screening of aquatic plants in the US, and may have additional utility for prioritizing management efforts of established species.