The photophysics of isolated protein chromophores

Gas-phase absorption properties of chromophores of several photoactive proteins have been studied experimentally at the electrostatic heavy-ion storage ring ELISA in Aarhus. The absorption wavelength has been calculated using an augmented effective Hamiltonian technique based on the multiconfigurational quasi-degenerate perturbation theory. The results have been compared to those of widely used state-specific second-order perturbation theory formalisms and their multistate extensions and also to ground-state linear response methods. It would appear that ab initio theory is now at a stage where the intrinsic properties of the chromophore molecules may be predicted with reasonable precision. There is evidence that in terms of absorption there is almost vacuum-like conditions in the hydrophobic interior of some proteins like the green fluorescent protein (GFP). In others, like for example the visual opsins, some significant perturbations are responsible for colour tuning.     

Interactions of like-charged rods at low temperatures: Analytical theory vs. simulations

We investigate a system consisting of two like-charged infinitely long rods and neutralizing counterions at low temperatures, using both analytic theory and simulations. With some reasonable approximations we can analytically solve for several ground-state structures of the model, starting with states where all counterions are lined up in the gap between the rods, over planar configurations, where the counterions are divided up into a fraction which resides between the rods, and counterions which are located on the outer surfaces, up to configurations which cover the full rod surfaces. Using parallel tempering simulations, we are able to study the system over a wide range of temperatures. At low temperatures we find good agreement with our T = 0 results. At higher temperatures, the strong coupling (SC) theory delivers qualitatively better results. We furthermore demonstrate that for the SC theory and our ground-state approximations to yield quantitative agreement, three parameters are required to be large, the strong-coupling parameter Ξ, the Rouzina-Bloomfield parameter, and the ratio of the average distance of the counterions to the radius of the rods. In the case of the latter ratio being small, our T = 0 results show better agreement with the simulation data at very low temperatures.     

Quantitation of bacteria through adsorption of intracellular biomolecules on carbon paste and screen-printed carbon electrodes and voltammetry of redox-active probes

A new electrochemical method for the quantitation of bacteria that is rapid, inexpensive, and amenable to miniaturization is reported. Cyclic voltammetry was used to quantitate M. luteus, C. sporogenes, and E. coli JM105 in exponential and stationary phases, following exposure of screen-printed carbon working electrodes (SPCEs) to lysed culture samples. Ferricyanide was used as a probe. The detection limits (3s) were calculated and the dynamic ranges for E. coli (exponential and stationary phases), M. luteus (exponential and stationary phases), and C. sporogenes (exponential phase) lysed by lysozyme were 3 × 10⁴ to 5 × 10⁶ colony-forming units (CFU) mL⁻¹, 5 × 10⁶ to 2 × 10⁸ CFU mL⁻¹ and 3 × 10³ to 3 × 10⁵ CFU mL⁻¹, respectively. Good overlap was obtained between the calibration curves when the electrochemical signal was plotted against the dry bacterial weight, or between the protein concentration in the bacterial lysate. In contrast, unlysed bacteria did not change the electrochemical signal of ferricyanide. The results indicate that the reduction of the electrochemical signal in the presence of the lysate is mainly due to the fouling of the electrode by proteins. Similar results were obtained with carbon-paste electrodes although detection limits were better with SPCEs. The method described herein was applied to quantitation of bacteria in a cooling tower water sample. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Activated dynamics of semiflexible polymers on structured substrates

We study the thermally activated motion of semiflexible polymers in double-well potentials using field-theoretic methods. Shape, energy, and effective diffusion constant of kink excitations are calculated, and their dependence on the bending rigidity of the semiflexible polymer is determined. For symmetric potentials, the kink motion is purely diffusive whereas kink motion becomes directed in the presence of a driving force. We determine the average velocity of the semiflexible polymer based on the kink dynamics. The Kramers escape over the potential barriers proceeds by nucleation and diffusive motion of kink-antikink pairs, the relaxation to the straight configuration by annihilation of kink-antikink pairs. We consider both uniform and point-like driving forces. For the case of point-like forces the polymer crosses the potential barrier only if the force exceeds a critical value. Our results apply to the activated motion of biopolymers such as DNA and actin filaments or of synthetic polyelectrolytes on structured substrates.     

A novel type of matrix for surface-assisted laser desorption–ionization mass spectrometric detection of biomolecules using metal-organic frameworks

A 3D metal-organic framework (MOF) nanomaterial as matrix for surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) and tandem mass spectrometry (MS/MS) was developed for the analysis of complex biomolecules. Unlike other nanoparticle matrices, this MOF nanomaterial does not need chemical modification prior to use. An exceptional signal reproducibility as well as very low background interferences in analyzing mono-/di-saccharides, peptides and complex starch digests demonstrate its high potential for biomolecule assays, especially for small molecules.     

Photophysics, photochemistry and photobiology of curcumin: Studies from organic solutions, bio-mimetics and living cells

Curcumin, with its recent success as an anti-tumor agent, has been attracting researchers from wide ranging fields of physics, chemistry, biology and medicine. The chemical structure of curcumin has two o-methoxy phenols attached symmetrically through α,β-unsaturated β-diketone linker, which also induces keto–enol tautomerism. Due to this, curcumin exhibits many interesting photophysical and photochemical properties. The absorption maximum of curcumin is 408–430 nm in most of the organic solvents, while the emission maximum is very sensitive to the surrounding solvent medium (460–560 nm) and the Stokes’ shift varied from 2000 to 6000 cm⁻¹. The fluorescence quantum yield in most of the solvents is low and reduced significantly in presence of water. The fluorescence lifetime is short (<1 ns) and displayed multi-exponential decay profile. The singlet excited states of curcumin decay by non-radiative processes contributed mainly by intra- and intermolecular proton transfer with very low intersystem crossing efficiency. Polarity, π-bonding nature, hydrogen bond donating and accepting properties of the solvent influence the excited state photophysics of curcumin in a complex manner. The triplet excited states of curcumin absorb at 720 nm and react with oxygen to produce singlet molecular oxygen. The photodegradation of curcumin produces smaller phenols and the photobiological activity of curcumin is due to the generation of reactive oxygen species.Being lipophilic in nature, the water solubility of curcumin could be enhanced upon the addition of surfactants, polymers, cyclodextrins, lipids and proteins. Changes in the absorption and fluorescence properties of curcumin have been found useful to follow its interaction and site of binding in these systems. Curcumin fluorescence could be employed to follow the unfolding pattern and structural changes in proteins. The intracellular curcumin showed more fluorescence in tumor cells than in normal cells and fluorescence spectroscopy could be used to monitor its preferential localization in the membrane of tumor cells. This review, presents the current status of research on the photophysical, photochemical and photobiological processes of curcumin in homogeneous solutions, bio-mimetics and living cells. Based on these studies, the possibility of developing curcumin, as a bimolecular sensitive fluorescent probe is also discussed.     

New accessory for studies of isotopic 1H/2H exchange and biomolecular interactions using transmission infrared spectroscopy

We present here a new accessory for IR transmission measurements of ¹H/²H exchange, as an ancillary tool for monitoring structural features of biomolecules in aqueous solution. This new accessory results from the combination of two dialysis membranes and a conventional liquid cell having two cylinders containing ²H₂O buffer. When compared with conventional transmission measurements, carried out either after dissolving lyophilized biomolecules in ²H₂O or after dialyzing the aqueous solution considered against ²H₂O buffer, this accessory shows the following advantages: (1) controlled measurements over the initial steps of this isotopic exchange and absence of molecular aggregation, and (2) smaller sample amounts. This new Fourier transform IR cell can also be used to analyze ligand–biomolecule and drug–cell interactions.      

New chromogenic and fluorogenic reagents and sensors for neutral and ionic analytes based on covalent bond formation–a review of recent developments

To date, hydrogen bonding and Coulomb, van der Waals and hydrophobic interactions are the major contributors to non-covalent analyte recognition using ionophores, ligands, aptamers and chemosensors. However, this article describes recent developments in the use of (reversible) covalent bond formation to detect analyte molecules, with special focus on optical signal transduction. Several new indicator dyes for analytes such as amines and diamines, amino acids, cyanide, formaldehyde, hydrogen peroxide, organophosphates, nitrogen oxide and nitrite, peptides and proteins, as well as saccharides have become available. New means of converting analyte recognition into optical signals have also been introduced, such as colour changes of chiral nematic layers. This article gives an overview of recent developments and discusses response mechanisms, selectivity and sensitivity.     

SPM analysis of fibrinogen adsorption on solid surfaces

The adsorption kinetics, adhesion and orientation of human fibrinogen on solid surfaces have been studied by surface probe microscopy (SPM) and quartz crystal microbalance techniques (QCM). CF₃-, NH₂-terminated organo-silane self-assembled monolayers (SAM) and OH-terminated silicon dioxide have been used as model surfaces. Furthermore, the interaction of fibrinogen with nanocomposite Ti/hydrocarbon plasma polymer films (Ti/ppCH) deposited by dc magnetron sputtering has also been studied.