Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins.

iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin's C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin's N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation results in a slight change in absorption and a 4-8 nm red shift in the fluorescence relative to iLOV, in good agreement with the computational predictions. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red shifting may be achieved by bringing the side chain closer to the cofactor.

The effect of hydrogen-bonding on flavin's infrared absorption spectrum.

Cluster and continuum solvation computational models are employed to model the effect of hydrogen bonding interactions on the vibrational modes of lumiflavin. Calculated spectra were compared to experimental Fourier-transform infrared (FTIR) spectra in the diagnostic 1450-1800 cm-1 range, where intense νC=C, νC=N, [Formula: see text] , and [Formula: see text] stretching modes of flavin's isoalloxazine ring are found. Local mode analysis is used to describe the strength of hydrogen-bonding in cluster models. The computations indicate that νC=C and νC=N mode frequencies are relatively insensitive to intermolecular interactions while the [Formula: see text] and [Formula: see text] modes are sensitive to direct (and also indirect for [Formula: see text] ) hydrogen-bonding interactions. Although flavin is neutral, basis sets without the diffuse functions provide incorrect relative frequencies and intensities. The 6-31+G* basis set is found to be adequate for this system, and there is limited benefit to considering larger basis sets. Calculated vibrational mode frequencies agree with experimentally determined frequencies in solution when cluster models with multiple water molecules are used. Accurate simulation of relative FTIR band intensities, on the other hand, requires a continuum (or possibly quantum mechanical/molecular mechanical) model that accounts for long-range electrostatic effects. Finally, an experimental peak at ca. 1624 cm-1 that is typically assigned to the [Formula: see text] vibrational stretching mode has a complicated shape that suggests multiple underlying contributions. Our calculations show that this band has contributions from both the C6-C7 and C2 = O stretching vibrations.

Electronic spectra of flavin in different redox and protonation states: a computational perspective on the effect of the electrostatic environment.

Flavins are versatile molecules due to their ability to exist in multiple redox and protonation states. At physiological conditions, they are usually encountered either as oxidized quinones, neutral semiquinones, anionic semiquinones, neutral hydroquinones, or anionic hydroquinones. We compute the electronic near-UV/vis spectra for flavin in each of these five states. Specifically, we compute vertical, adiabatic, and vibronic excitations for all excited states that have wavelengths longer than 300 nm. We employ the calculations to assign the peaks in the corresponding experimental UV/vis spectra from literature. We also compare the effect of polar and non-polar solvents on the spectra using a polarizable continuum model. Finally, we construct "electrostatic spectral tuning maps" for prominent peaks in each of the five states. These maps qualitatively describe how the flavin electronic spectra will be shifted by an anisotropic electrostatic environment such as a protein. Understanding how flavin's UV/vis absorption spectrum is modulated by its environment can aid in experiments employing flavin as a probe of internal electrostatics of a protein and in engineering new color variants of flavoproteins.

Electrostatic Spectral Tuning Maps for Biological Chromophores.

The mechanism by which the absorption wavelength of a molecule is modified by a protein is known as spectral tuning. Spectral tuning is often achieved by electrostatic interactions that stabilize/destabilize or modify the shape of the excited and ground-state potential energy surfaces of the chromophore. We present a protocol for the construction of three-dimensional "electrostatic spectral tuning maps" that describe how vertical excitation energies in a chromophore are influenced by nearby charges. The maps are built by moving a charge on the van der Waals surface of the chromophore and calculating the change in its excitation energy. The maps are useful guides for protein engineering of color variants, for interpreting spectra of chromophores that act as probes of their environment, and as starting points for further quantum mechanical/molecular mechanical studies. The maps are semiquantitative and can approximate the magnitude of the spectral shift induced by a point charge at a given position with respect to the chromophore. We generate and discuss electrostatic spectral tuning maps for model chromophores of photoreceptor proteins, fluorescent proteins, and aromatic amino acids. Such maps may be extended to other properties such as oscillator strengths, absolute energies (stability), ionization energies, and electron affinities.

Fluorescence Properties of Flavin Semiquinone Radicals in Nitronate Monooxygenase.

Fluorescent cofactors like flavins can be exploited to probe their local environment with spatial and temporal resolution. Although the fluorescence properties of the oxidized and two-electron-reduced states of flavins have been studied extensively, this is not the case for the one-electron-reduced state. Both the neutral and anionic semiquinones have proven particularly challenging to examine, as they are unstable in solution and are transient, short-lived species in many catalytic cycles. Here, we report that the nitronate monooxygenase (NMO) from Pseudomonas aeruginosa PAO1 is capable of stabilizing both semiquinone forms anaerobically for hours, thus enabling us to study their spectroscopy in a constant protein environment. We found that in the active site of NMO, the anionic semiquinone exhibits no fluorescence, whereas the neutral semiquinone radical shows a relatively strong fluorescence, with a behavior that violates the Kasha-Vavilov rule. These fluorescence properties are discussed in the context of time-dependent density functional theory calculations, which reveal low-lying dark states in both systems.

Cationic Metallo-Polyelectrolytes for Robust Alkaline Anion-Exchange Membranes.

Chemically inert, mechanically tough, cationic metallo-polyelectrolytes were conceptualized and designed as durable anion-exchange membranes (AEMs). Ring-opening metathesis polymerization (ROMP) of cobaltocenium-containing cyclooctene with triazole as the only linker group, followed by backbone hydrogenation, led to a new class of AEMs with a polyethylene-like framework and alkaline-stable cobaltocenium cation for ion transport. These AEMs exhibited excellent thermal, chemical and mechanical stability, as well as high ion conductivity.

Binding of Cobaltocenium-containing Polyelectrolytes with Anionic Probes.

Cationic cobaltocenium-containing polyelectrolytes have a unique ability to form ionic complex with various anionic species. We carried out two sets of model study to compare the relative binding strength of a cobaltocenium-containing polyelectrolyte. First, the nature and relative strength of intermolecular interaction between cobaltocenium-containing polyelectrolytes and different anionic probes were investigated by spectroscopic methods. A dye-displacement method was used to monitor absorbance and fluorescence emissions. Second, the binding strength of this cobaltocenium-containing polyelectrolyte was compared with a classical quaternary ammonium polymer. Formation of polyelectrolyte complex between the cobaltocenium-containing polyelectrolyte and a common anionic polyelectrolyte at various concentrations was examined by optical absorption and light scattering.

Metallocene-Containing Homopolymers and Heterobimetallic Block Copolymers via Photoinduced RAFT Polymerization.

We report the synthesis of cationic cobaltocenium and neutral ferrocene containing homopolymers mediated by photoinduced reversible addition-fragmentation chain transfer (RAFT) polymerization with a photocatalyst fac-[Ir(ppy)3]. The homopolymers were further used as macromolecular chain transfer agents to synthesize diblock copolymers via chain extension. Controlled/"living" feature of photoinduced RAFT polymerization was confirmed by kinetic studies even without prior deoxygenation. A light switch between ON and OFF provided a spatiotemporal control of polymerization.