Extending the range of FRET—the Monte Carlo study of the antenna effect

The problem of extending the utilizable range of Förster resonance energy transfer (FRET) is of great current interest, due to the demand of conformation studies of larger biological structures at distances exceeding typical limiting distance of 100 Å. One of the ways to address this issue is the use of so-called antenna effect. In the present work, the influence of the antenna effect on the FRET efficiency is investigated by the Monte Carlo analysis. The previously published results Bojarski et al. (J Phys Chem B 115:10120–10125, 2011) indicate that using a simple model of donor linked with a protein labeled with multiple acceptors, significantly increases the transfer efficiency in comparison with donor–single acceptor system. The effect is stronger if the transition moments of acceptors are mutually parallel. In this work, to extend the scope of possible biological systems to be analyzed, different distributions of donor–acceptors distance are analyzed, as well as the size and shape of the attached molecule

Interpreting resonance energy transfer experiments with monte-carlo and molecular dynamic simulations

Forster resonance energy transfer (FRET) is a photophysical process in which an electronically excited chromophore spontaneously transfers energy to another molecule by a non-radiative dipole-dipole interaction. While this occurs naturally in processes such as light harvesting in photosynthetic organisms, it is now commonly used as a tool in molecular biophysics to examine the proximity and structure of biological macromolecules and to report on biochemical events. One of the features making FRET so widely used is the strong dependence of transfer on the distance between the participating molecules. Due to this, it has been termed a {u0300}spectroscopic ruler' enabling the measurement of intermolecular distances and conformations of proteins and nucleic acids. Although the utility of this method is now well appreciated, many difficulties arise in interpreting FRET experiments to gain detailed quantitative information.One complication is that the chance of transfer taking place is dependent on the orientation of the participating molecules, something that is difficult (if not impossible) to measure. Another difficulty comes from the fact that the commonly used fluorescent dyes are characterised by high conformational flexibility, which allows them to sample substantial space around the point of attachment introducing further uncertainty to the distance measurements. Moreover, the range of possible application of FRET as a spectroscopic ruler is limited to 10 nm, whereas many of biological events that could potentially be signalled by FRET occur on larger distances. In my work, I aim to use computational techniques to address these difficulties, by developing tools that can be used to better interpret the results of FRET experiments and to contribute to the design of new experiments employing FRET

Accounting for dye diffusion and orientation when relating FRET measurements to distances: three simple computational methods

Förster resonance energy transfer (FRET) allows in principal for the structural changes of biological systems to be revealed by monitoring distributions and distance fluctuations between parts of individual molecules. However, because flexible probes us

Do bifunctional labels solve the problem of dye diffusion in FRET analysis?

We examine the potential application of bifunctional dyes in Förster resonance energy transfer (FRET) experiments due to their increasing popularity in electron paramagnetic resonance spectroscopy. To do this we conduct molecular simulations of the wel

Identification of a new Kir6 potassium channel and comparison of properties of Kir6 subtypes by structural modelling and molecular dynamics

ATP-sensitive potassium ion channels (KATP) are transmembrane proteins that modulate insulin release and muscle contraction. KATP channels are composed of two types of subunit, Kir6 and SUR, which exist in two and three isoforms respectively with different tissue distribution. In this work, we identify a previously undescribed ancestral vertebrate gene encoding a Kir6-related protein that we have named Kir6.3, which may not have a SUR binding partner, unlike the other two Kir6 proteins. Whereas Kir6.3 was lost in amniotes including mammals, it is still present in several early-diverging vertebrate lineages such as frogs, coelacanth, and rayfinned fishes. Molecular dynamics (MD) simulations using homology models of Kir6.1, Kir6.2, and Kir6.3 from the coelacanth Latimeria chalumnae showed that the three proteins exhibit subtle differences in their dynamics. Steered MD simulations of Kir6-SUR pairs suggest that Kir6.3 has a lower binding affinity for the SUR proteins than either Kir6.1 or Kir6.2. As we found no additional SUR gene in the genomes of the species that have Kir6.3, it most likely forms a lone tetramer. These findings invite studies of the tissue distribution of Kir6.3 in relation to the other Kir6 as well as SUR proteins to determine the functional roles of Kir6.3

Enhancing the Inhomogeneonus Photodynamics of Canonical Bacteriophytochrome

The ability of phytochromes to act as photoswitches in plants and microorganisms depends on interactions between a bilin-like chromophore and a protein. The interconversion occurs between the spectrally distinct red (Pr) and far-red (Pfr) conformers. This conformational change is triggered by the photoisomerization of the chromophore D-ring pyrrole. In this study, as a representative example of a phytochrome-bilin system, we take biliverdin IXα (BV) bound to bacteriophytochrome (BphP) from Deinococcus radiodurans. In the absence of light, we use an enhanced sampling molecular dynamics (MD) method to overcome the photoisomerization energy barrier. We find that the calculated free energy (FE) barriers between essential metastable states agree with spectroscopic results. We show that the enhanced dynamics of the BV chromophore in BphP triggers nanometer-scale conformational movements that propagate by two experimentally determined signal transduction pathways. Most importantly, we describe how the metastable states enable a thermal transition known as the dark reversion between Pfr and Pr, through a previously unknown intermediate state of Pfr. Here, for the first time, the heterogeneity of temperature-dependent Pfr states is presented at the atomistic level. This work paves a way toward understanding the complete mechanism of the photoisomerization of a bilin-like chromophore in phytochromes

Comparing the Ability of Enhanced Sampling Molecular Dynamics Methods to Reproduce the Behavior of Fluorescent Labels on Proteins

Adequately sampling the large number of conformations accessible to proteins and other macromolecules is one of the central challenges in molecular dynamics (MD) simulations; this activity can be difficult, even for relatively simple systems. An example where this problem arises is in the simulation of dye-labeled proteins, which are now being widely used in the design and interpretation of Förster resonance energy transfer (FRET) experiments. In this study, MD simulations are used to characterize the motion of two commonly used FRET dyes attached to an immobilized chain of polyproline. Even in this simple system, the dyes exhibit complex behavior that is a mixture of fast and slow motions. Consequently, very long MD simulations are required to sufficiently sample the entire range of dye motion. Here, we compare the ability of enhanced sampling methods to reproduce the behavior of fluorescent labels on proteins. In particular, we compared Accelerated Molecular Dynamics (AMD), metadynamics, Replica Exchange Molecular Dynamics (REMD), and High Temperature Molecular Dynamics (HTMD) to equilibrium MD simulations. We find that, in our system, all of these methods improve the sampling of the dye motion, but the most significant improvement is achieved using REMD