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 usually have to be attached to the macromolecule to conduct these experiments, they suffer from uncertainty in probe positions and orientations. One of the way to address this issue is to use molecular dynamics simulations to explicitly model the likely positions of the probes, but, this is still not widely accessible because of the large computational effort required. Here we compare three simpler methods that can potentially replace MD simulations in FRET data interpretation. In the first, the volume accessible for dye movement is calculated using a fast, geometrical algorithm. The next method, adapted from the analysis of electron paramagnetic studies, utilises a library of rotamers describing probe conformations. The last method uses preliminary MD simulations of fluorescent dyes in solution, to identify all conformational states of dyes and overlays this on the macromolecular system. A comparison of these methods in the simple system of dye-labelled polyproline, shows that in the case of lack of interaction between the dye and host, all give results comparable with MD simulations but require much less time. Differences between these three methods and their ability to compete with MD simulations in the analysis of real experiment are demonstrated and discussed using the examples of cold shock protein and leucine transporter systems.

A new method for extending the utilizable range of Förster resonance energy transfer (FRET) is proposed and tested by the Monte Carlo technique. The obtained results indicate that the efficiency of FRET can be significantly enhanced at a given distance if the energy transfer takes place toward multiple acceptors that are closely located on a macromolecule instead of a single acceptor molecule as it is currently used in FRET analysis. On the other hand, reasonable FRET efficiency can be obtained at significantly longer distances than in the case of a single acceptor. Randomly distributed and parallel orientated acceptor transition moments with respect to the transition moment of the donor molecule have been analyzed as two extreme cases. As expected, a parallel orientation of donor and acceptor transition moments results in a more efficient excitation energy transfer. This finding could be used to directly reveal the assembly/deassembly of large protein complexes in a cell by fluorescence microscopy.

Inward rectifying potassium ion channels (KATP), sensitive to the ATP/ADP concentration ratio, play an important, control role in pancreatic β cells. The channels close upon the increase of this ratio, which, in turn, triggers insulin release to blood. Numerous mutations in KATP lead to severe and widespread medical conditions such as diabetes. The KATP system consists of a pore made of four Kir6.2 subunits and four accompanying large SUR1 proteins belonging to the ABCC transporters group. How SUR1 affects KATP function is not yet known; therefore, we created simplified models of the Kir6.2 tetramer based on recently determined cryo-EM KATP structures. Using all-atom molecular dynamics (MD) with the CHARMM36 force field, targeted MD, and molecular docking, we revealed functionally important rearrangements in the Kir6.2 pore, induced by the presence of the SUR1 protein. The cytoplasmic domain of Kir6.2 (CTD) is brought closer to the membrane due to interactions with SUR1. Each Kir6.2 subunit has a conserved, functionally important, disordered N-terminal tail. Using molecular docking, we found that the Kir6.2 tail easily docks to the sulfonylurea drug binding region located in the adjacent SUR1 protein. We reveal, for the first time, dynamical behavior of the Kir6.2/SUR1 system, confirming a physiological role of the Kir6.2 disordered tail, and we indicate structural determinants of KATP-dependent insulin release from pancreatic β cells.