Testing the use of molecular dynamics to simulate fluorophore motions and FRET

Fluorescence resonance energy transfer (FRET) is commonly used to determine the proximity of fluorophores, but usually many assumptions are required to gain a quantitative relationship between the likelihood of energy transfer and fluorophore separation. Molecular Dynamics (MD) simulations provide one way of checking these assumptions, but before using simulations to study complex systems it is important to make sure that they can correctly model the motions of fluorophores and the likely FRET efficiency in a simple system. Here we simulate a well characterised situation of independent fluorophores in solution so that we can compare the predictions with expected values. Our simulations reproduce the experimental fluorescence anisotropy of Alexafluor488 and predict that of AlexaFluor568. At the ensemble level we are able to reproduce the expected isotropic and dynamic motion of the fluorophores as well as the FRET efficiency of the system. At the level of single donor-acceptor pairs, however, very long simulations are required to adequately sample the translational motion of the fluorophores and more surprisingly also the rotational motion. Our studies demonstrate how MD simulations can be used in more complex systems to check if the dynamic orientation averaging regime applies, if the fluorophores have isotropic orientational motion, to calculate the likely values of the orientation factor κ(2) and to determine the FRET efficiency of the system in both dynamic and static orientational averaging regimes. We also show that it is possible in some situations to create system specific relationships between FRET efficiency and fluorophore separation that can be used to interpret experimental data and find any correlations between κ(2) and separation that may influence distance measurements.     

Screening of a minimal enriched P450 BM3 mutant library for hydroxylation of cyclic and acyclic alkanes

A minimal enriched P450 BM3 library was screened for the ability to oxidize inert cyclic and acyclic alkanes. The F87A/A328V mutant was found to effectively hydroxylate cyclooctane, cyclodecane and cyclododecane. F87V/A328F with high activity towards cyclooctane hydroxylated acyclic n-octane to 2-(R)-octanol (46% ee) with high regioselectivity (92%).     

Collisions of Vortex Filament Pairs

We consider the problem of collisions of vortex filaments for a model introduced by Klein et al. (J Fluid Mech 288:201–248, 1995) and Zakharov (Sov Phys Usp 31(7):672–674, 1988, Lect. Notes Phys 536:369–385, 1999) to describe the interaction of almost parallel vortex filaments in three-dimensional fluids. Since the results of Crow (AIAA J 8:2172–2179, 1970) examples of collisions are searched as perturbations of antiparallel translating pairs of filaments, with initial perturbations related to the unstable mode of the linearized problem; most results are numerical calculations. In this article, we first consider a related model for the evolution of pairs of filaments, and we display another type of initial perturbation leading to collision in finite time. Moreover, we give numerical evidence that it also leads to collision through the initial model. We finally study the self-similar solutions of the model.     

Highly Enantioselective Rhodium(I)‐Catalyzed Activation of Enantiotopic Cyclobutanone C?C Bonds

The selective functionalization of carbon–carbon σ bonds is a synthetic strategy that offers uncommon retrosynthetic disconnections. Despite progress in C C activation and its great importance, the development of asymmetric reactions lags behind. Rhodium(I)‐catalyzed selective oxidative additions into enantiotopic C C bonds in cyclobutanones are reported. Even operating at a reaction temperature of 130 °C, the process is characterized by outstanding enantioselectivity with the e.r. generally greater than 99.5:0.5. The intermediate rhodacycle is shown to react with a wide variety of tethered olefins to deliver complex bicyclic ketones in high yields.     

Hydrophilic properties as a new contribution for computer-aided identification of short peptides in complex mixtures

A new method to predict elementary amino acid (AA) composition of peptides (molar mass <1,000 g/mol) is described. This procedure is based on a computer-aided method using three combined analyses-reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and capillary electrophoresis coupled with mass spectrometry-and using a software calculating all possible amino acid combinations from the mass of any given peptide. The complementarity between HILIC and RPLC was demonstrated. Peptide retention prediction in HILIC was successfully modelled, and the achieved prediction accuracy was as high as r2=0.97. This mathematical model, based on amino acid retention contributions and peptide length, provided the information about peptide hydrophilicity that was not redundant with its hydrophobicity. Correlations between respectively the hydrophobicity coefficients and RPLC retention time, hydrophilicity and HILIC retention time, and electrophoretic mobility and migration time were used for ranking all potential AA combinations corresponding to the given mass. The essential contribution of HILIC in this identification strategy and the need to combine the three models to significantly increase identification capabilities were both shown. Applied to an 18-standard peptide mixture, the identification procedure enabled the actual AA combination determination of the 14 di- to pentapeptides, in addition to an over 98 % reduction of possible combination numbers for the four hexapeptides. This procedure was then applied to the identification of 24 unknown peptides in a rapeseed protein hydrolysate. The effective AA composition was found for ten peptides, whereas for the 14 other peptides, the number of possible combinations was reduced by over 95 % thanks to the association of the three analyses. Finally, as a result of the information provided by the analytical techniques about peptides present in the mixture, the proposed method could become a highly valuable tool to recover bioactive peptides from undefined protein hydrolysates.     

Mild and rational synthesis of palladium complexes comprising C(4)-bound N-heterocyclic carbenes

Oxidative addition of pyridyl-functionalised 4-iodoimidazolium salts to palladium(0) gives catalytically active complexes in which the N-heterocyclic carbene is bound to the palladium(II) centre in a non-classical bonding mode via C(4).     

5‐aminothiazolium salts as potential cyclic azomethine ylides: Base‐induced cycloaddition reactions with aryl,alkyl, and benzoyl isothiocyanates

Reactions of the in situ generated thiazoles 2 with aryl and alkyl isothiocyanates appear to be totally regioselective and give the unexpected 5‐(phenylthio)imidazolium‐4‐thiolates 3 . Such rapid interconversion of mesoionic compounds is explained by a 1,3‐dipolar addition to the C=N bond of the heterocumulene followed by tBuNCS elimination. Similar interactions with benzoyl isothiocyanate exclusively proceed on the C=S unsaturation of the heteroallene moiety and produce the 4‐(phenylthio)thiazolium‐5‐amidines 12 . Structural assignment of isolated imidazoles and thiazoles is based on ¹³C NMR data and chemically confirmed by the NaBH₄ reduction of the alkylated derivatives 5 and 13 . Efforts to isomerize the starting mesoionic thiazole 2a without the use of tBuNCS are unsuccessful. © 2000 John Wiley & Sons, Inc. Heteroatom Chem 10:16–26, 2000