Molecular Gating of an Engineered Enzyme Captured in Real Time.

Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessibility of their active sites by molecular gates. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, single-molecule fluorescence spectroscopy, and molecular dynamics. Photoinduced electron-transfer-fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at the single-molecule level with rate constants ( kon = 1822 s-1, koff = 60 s-1) corresponding well with those from the pre-steady-state kinetics ( k-1 = 1100 s-1, k1 = 20 s-1). The PET-FCS technique is used here to study the conformational dynamics in a soluble enzyme, thus demonstrating an additional application for this method. Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts.

Tuning excited-state proton transfer dynamics of a 3-hydroxychromone dye in supramolecular complexes via host-guest steric compatibility.

The photophysics of 2-(2'-benzofuryl)-3-hydroxychromone (BFHC) is remarkably modulated in its complexes with macrocyclic hosts such as ß-cyclodextrin (ß-CD), hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and methyl-ß-cyclodextrin (M-ß-CD). BFHC exhibits dual emission bands, attributable to excited normal (N*) and tautomer (T*) forms, where the latter originates from the former through an excited-state intramolecular proton transfer (ESIPT) reaction. Fluorescence lifetimes of the tautomer, along with the intensity ratio (IT*/IN*) of the dual emission bands, and the fluorescence quantum yield (Φ) of the dye, increase significantly in the order ß-CD < HP-ß-CD < M-ß-CD to indicate increasing hydrophobicity of the dye environment in the host CD cavity. In accordance with this increasing hydrophobicity of the dye environment, the ESIPT dynamics of BFHC becomes increasingly fast in the host cavity in the order ß-CD < HP-ß-CD < M-ß-CD. Binding constant data and molecular modeling studies indicate that the increasing order of the faster ESIPT dynamics originates from an increasingly tight host-guest spatial fit, which causes increasingly strong dehydration of the BFHC dye. Steric compatibility in size and shape between the host cavity and the guest, which modulates the tightness of the host-guest spatial fit and hence the extent of hydration, is a key factor for tuning the proton transfer dynamics since water molecules perturb the ESIPT reaction and quench the fluorescence of BFHC.

6,7-dimethoxy-coumarin as a probe of hydration dynamics in biologically relevant systems.

Coumarin derivatives are well known fluorescence reporters for investigating biological systems due to their strong micro-environment sensitivity. Despite having wide range of environment sensitive fluorescence probes, the potential of 6,7-dimethoxy-coumarin has not been studied extensively so far. With a perspective of its use in protein studies, namely using the unnatural amino acid technology or as a substrate for hydrolase enzymes, we study acetyloxymethyl-6,7-dimethoxycoumarin (Ac-DMC). We investigate the photophysics and hydration dynamics of this dye in aerosol-OT (AOT) reverse micelles at various water contents using the time dependent fluorescence shift (TDFS) method. The TDFS response in AOT reverse micelles from water/surfactant ratio of 0 to 20 confirms its sensitivity towards the hydration and mobility of its microenvironment. Moreover, we show that the fluorophore can be efficiently quenched by halide ions. Hence, we conclude that the 6,7-dimethoxy-methylcoumarin fluorophore is useful for studying hydration parameters in biologically relevant systems.

Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive Fluorescence Probes for Molecular and Cellular Applications.

In this work, we used firefly oxyluciferin (OxyLH2) and its polarity-dependent fluorescence mechanism as a sensitive tool to monitor biomolecular interactions. The chromophores, OxyLH2, and its two analogues, 4-MeOxyLH and 4,6'-DMeOxyL, were modified trough carboxylic functionalization and then coupled to the N-terminus part of Tat and NCp7 peptides of human immunodeficiency virus type-1 (HIV-1). The photophysical properties of the labeled peptides were studied in live cells as well as in complex with different oligonucleotides in solution. By monitoring the emission properties of these derivatives we were able, for the first time, to study in vitro biomolecular interactions using oxyluciferin as a sensor. As an additional application, cyclopropyl-oxyluciferin (5,5-Cpr-OxyLH) was site-specifically conjugated to the thiol group (Cys-232) of the human protein α-1 antytripsin to investigate its interaction with porcine pancreatic elastase. Our data demonstrate that OxyLH2 and its derivatives can be used as fluorescence reporters for monitoring biomolecular interactions.

Emission properties of oxyluciferin and its derivatives in water: revealing the nature of the emissive species in firefly bioluminescence.

The first systematic steady-state and time-resolved emission study of firefly oxyluciferin (emitter in firefly bioluminescence) and its analogues in aqueous buffers provided the individual emission spectra of all chemical forms of the emitter and the excited-state equilibrium constants in strongly polar environment with strong hydrogen bonding potential. The results confirmed the earlier hypothesis that excited-state proton transfer from the enol group is favored over proton transfer from the phenol group. In water, the phenol-keto form is the strongest photoacid among the isomers and its conjugate base (phenolate-keto) has the lowest emission energy (634 nm). Furthermore, for the first time we observed green emission (525 nm) from a neutral phenol-keto isomer constrained to the keto form by cyclopropyl substitution. The order of emission energies indicates that in aqueous solution a second deprotonation at the phenol group after the enol group had dissociated (that is, deprotonation of the phenol-enolate) does not occur in the first excited state. The pH-dependent emission spectra and the time-resolved fluorescence parameters revealed that the keto-enol tautomerism reaction, which can occur in a nonpolar environment (toluene) in the presence of a base, is not favored in water.