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).

Electrical unfolding of cytochrome 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.

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.

Oxygen-Induced In Situ Manipulation of the Interlayer Coupling and Exciton Recombination in BiSe/MoS 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 BiSe/MoS 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.

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 ( T), is nearly monoexponential and displays a weak temperature dependence. Below T, 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.

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 (∼10 s) 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 (∼10 s). 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.

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.

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).

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.

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.

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%).

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.

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.

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.

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.

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.

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).

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.

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.

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.

Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA.

Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated.

Vibrational coherence spectroscopy of the heme domain in the CO-sensing transcriptional activator CooA.

Femtosecond vibrational coherence spectroscopy was used to investigate the low-frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (k(B)T = 200 cm(-1)) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods.

Spectroscopic identification of reactive porphyrin motions.

Nuclear resonance vibrational spectroscopy (NRVS) reveals the vibrational dynamics of a Mössbauer probe nucleus. Here, (57)Fe NRVS measurements yield the complete spectrum of Fe vibrations in halide complexes of iron porphyrins. Iron porphine serves as a useful symmetric model for the more complex spectrum of asymmetric heme molecules that contribute to numerous essential biological processes.

Investigation of the low frequency dynamics of heme proteins: native and mutant cytochrome P450(cam) and redox partner complexes.

Vibrational coherence spectroscopy (VCS) is used to investigate the low-frequency dynamics of camphor-free and camphor-bound cytochrome P450(cam) (CYP 101) and its L358P mutant. The low-frequency heme vibrations are found to be perturbed upon binding to the electron transfer partner putidaredoxin (Pdx). A strong correlation between the "detuned" vibrational coherence spectrum, which monitors frequencies between 100 and 400 cm(-1), and the lower frequency part of the Raman spectrum is also demonstrated.

Investigations of low-frequency vibrational dynamics and ligand binding kinetics of cystathionine beta-synthase.

Vibrational coherence spectroscopy is used to study the low frequency dynamics of the truncated dimer of human cystathionine beta-synthase (CBS). CBS is a pyridoxal-5'-phosphate-dependent heme enzyme with cysteine and histidine axial ligands that catalyzes the condensation of serine and homocysteine to form cystathionine. A strong correlation between the "detuned" coherence spectrum (which probes higher frequencies) and the Raman spectrum is demonstrated, and a rich pattern of modes below 200 cm(-1) is revealed.