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.
ATP-sensitive potassium
(KATP) channels are present in numerous
organs, including the heart, brain, and pancreas. Physiological opening
and closing of KATPs present in pancreatic β-cells, in response
to changes in the ATP/ADP concentration ratio, are correlated with
insulin release into the bloodstream. Sulfonylurea drugs, commonly
used in type 2 diabetes mellitus treatment, bind to the octamer KATP
channels composed of four pore-forming Kir6.2 and four SUR1 subunits
and increase the probability of insulin release. Azobenzene-based
derivatives of sulfonylureas, such as JB253 inspired by well-established
antidiabetic drug glimepiride, allow for control of this process by
light. The mechanism of that phenomenon was not known until now. In
this paper, we use molecular docking, molecular dynamics, and metadynamics
to reveal structural determinants explaining light-controlled insulin
release. We show that both
trans-
and
cis-
JB253 bind to the same SUR1 cavity as antidiabetic sulfonylurea glibenclamide
(GBM). Simulations indicate that, in contrast to
trans-
JB253, the
cis-
JB253 structure generated by blue
light absorption promotes open structures of SUR1, in close similarity
to the GBM effect. We postulate that in the open SUR1 structures,
the N-terminal tail from Kir6.2 protruding into the SUR1 pocket is
stabilized by flexible enough sulfonylureas. Therefore, the adjacent
Kir6.2 pore is more often closed, which in turn facilitates insulin
release. Thus, KATP conductance is regulated by peptide linkers between
its Kir6.2 and SUR1 subunits, a phenomenon present in other biological
signaling pathways. Our data explain the observed light-modulated
activity of photoactive sulfonylureas and widen a way to develop new
antidiabetic drugs having reduced adverse effects.
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.
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