| dc.description.abstracteng | Membrane channels are an essential part of any life form. They conduct
the selective flux across the cell membrane of many important
molecules that would otherwise not permeate. Experimental studies
on membrane channels have led to the structural and functional characterization
of many of them, yet many underlying physico-chemical
mechanisms are somewhat out of reach. The aim of this thesis is
to gain quantitative understanding on the structural and functional
properties of these proteins by means of computational methods, such
as Molecular Dynamics (MD) and free energy calculations. One of the
most common approaches to study the selectivity and permeation
mechanisms of a channel is the calculation of the Potential of Mean
Force (PMF) for solute permeation across the pore. Usually, PMFs are
calculated via MD simulations, which requires a significant amount of
computational power. Hence, we compared the capability of MD with
that of 3-Dimensional Reference Interaction-Site Model (3D-RISM), allegedly
as accurate as MD but much more computationally efficient, to
compute PMFs of solute permeation across Urea Transporter B (UT-B)
and Aquaporin 1 (AQP1). We found a remarkable agreement between
the PMFs for water permeation calculated from both techniques. However,
for the rest of tested solutes, namely ammonia, urea, molecular
oxygen, and methanol, we found critical discrepancies between
3D-RISM and with MD, which were found to be independent of the closure
relation, the choice of the reaction coordinate, or the fluctuations
of the protein. This suggests that, whilst 3D-RISM may provide reasonable
approximations on PMFs for the permeation of water, it is not
appropriate to study the permeation of uncharged non-water solutes.
We further investigated, via a combination of MD simulations and
free energy calculations, the structure and function of the fluoride-specific
channel Fluc-Bpe. The free energy calculations allowed us to
ascertain the specific nature of five isolated electron densities found
in the crystal structure of Fluc, four of which were provisionaly assigned
to fluoride, and the remaining one to sodium. We conducted
two different kinds of binding free energy calculations: i) relative binding
free energy differences ∆∆Gbind, and ii) absolute binding free energy
∆Gbind. Notably, the calculation of ∆∆Gbind allowed us to determine,
between two putative molecular species, namely water and
fluoride, which species was more likely to bind at a certain binding
site. The resulting free energies were partly dependent on fluoride-phenylalanine
interactions, which we found to be underestimated by
~ 30 kJ mol-1 in current additive force-fields. Thus, the disctimination
of one species over the other was only possible because the ∆∆Gbind. values largely deviated from zero. In turn, the calculation of ∆Gbind
allowed us to confirm whether a certain species would bind per se
to Fluc-Bpe. Besides, short, free MD simulations proved to be key to
assess the structural stability of the channel in different conditions,
which, together with the free energy calculations, indicated that the
four densities assigned to fluoride rather corresponded to ordered
water molecules, and that the last electron density corresponded to a
structural sodium.
We finally evaluated, using MD simulations, the response of Fluc-Bpe to the presence of fluoride ions restrained at the permeating pore.
The results suggested that the channel would undergo an opening
transition, after which water molecules enter the pore to solvate the
ions. Then, we calculated the PMFs for the permeation of water, fluoride
and chloride using Umbrella Sampling (US) simulations. The
profiles of solute permeation across the open structure indicated that
water, fluoride, chloride would efficiently permeate the channel, being
in stark contrast with the experimental evidence, which demonstrates
that Fluc channels permeate fluoride by a ~ 100-fold ratio over
chloride. We suspect that our results might be affected by the inaccurate
modelling of ion-protein contacts highlighted before. The proper
modelling of ion-protein interactions is extremely important for the
establishment of salt-bridges, the structural stability of proteins, or
the permeation of ions. Therefore, we conclude that our results regarding
the permeation mechanism in Fluc-Bpe mainly reflect the
imperfections of current additive force-fields, and that the usage of
polarizable force-fields and development of accurate ion-protein interactions
may certainly aid future research. | de |