Multiple pore lining residues modulate water permeability of GlpF

Abstract

The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin “open state” is still considered to be a continuously open pore with water molecules permeating in a single‐file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long‐term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications.

Quantification of Fundamental Weak Base and Ion Permeabilities in the Acidic pH Region Utilizing Conjugated Oregon Green in Liposome‐Based Assays

“…LUVs were subjected to a hyperosmotic solution (working buffer + 200 m m sucrose) in a stopped‐flow apparatus (SFM‐300 or m‐SFM, Bio‐Logic, Claix, France) at 277 or 295 K. The intensity of scattered light was monitored at 90° at a wavelength of 546 nm. [ 12,24,43 ] Water permeabilities ( P f ) through vesicles with a hyper osmotic gradient c s were calculated utilizing the analytical solution [ 23,44 ] $$$$\begin{eqnarray} V\ \left( t \right) = {V}_0\ \frac{{{c}_0}}{{{c}_0 + {c}_{\rm{s}}}}\left( {1 + L\left( {\frac{{{c}_{\rm{s}}}}{{{c}_0}}{\rm{exp}}\left( {\frac{{{c}_{\rm{s}}}}{{{c}_0}} - t \cdot \frac{{S{P}_{\rm{f}}{V}_{\rm{w}}{{\left( {{c}_0 + {c}_{\rm{s}}} \right)}}^2}}{{{V}_0{c}_0}}} \right)} \right)} \right)\nonumber\hspace*{-10pt}\\ \end{eqnarray}$$$$with inner vesicle osmolarity c 0 , Lambert function L , the surface area of lipid vesicles S , the molar volume of water V w , and V 0 the water volume at time t = 0. For estimating the volume V ( t ) the measured scattered light intensity ( I ( t )) was Taylor approximated in the form [ 44 ] $$$$\begin{equation}I\ \left( t \right) = a + bV\left( t \right) + dV{\left( t \right)}^2\end{equation}$$$$with …”

“…LUVs were subjected to a hyperosmotic solution (working buffer + 200 mm sucrose) in a stopped-flow apparatus (SFM-300 or m-SFM, Bio-Logic, Claix, France) at 277 or 295 K. The intensity of scattered light was monitored at 90°at a wavelength of 546 nm. [12,24,43] Water permeabilities (P f ) through vesicles with a hyper osmotic gradient c s were calculated utilizing the analytical solution [23,44]…”

“…As an example, with $$K_p = 2.2 \times 10^{-3}$$, [ 4 ] $${D}_{{\rm{NH}}_3} = 1.5 \times {10}^{ - 5}{\rm{\ cm}}^2{\rm{\ s}}^{ - 1}$$ [ 5 ] and h = 4.1 nm [ 6 ] at RT, $${P}_{{\rm{NH}}_3} = 805 \mu {\rm{m\ s}}^{ - 1}$$ according to Equation () for the polar lipid extract (PLE) of Escherichia coli , a widely used natural lipid mixture in in vitro studies. [ 7–12 ] However, despite $${P}_{{\rm{NH}}_3}$$ being orders of magnitude higher as compared to passive membrane permeabilites of H 2 O, glycerol or urea, [ 13 ] NH 3 permeability across lipid bilayers is facilitated by the highly conserved Amt/Mep/Rh superfamily of transmembrane proteins. [ 14 ] Trimeric ammonium transport proteins (Amt) are found in bacteria, plants, yeast, and animals.…”

Membrane and Lumen-Compartmentalized Polymersomes for Biocatalysis and Cell Mimics

“…Escherichia coli glycerol facilitator (GlpF) is a channel protein for selectively transporting sugar alcohols, demonstrating a high degree of stereoselectivity in this function. 88 Additionally, they incorpo- rated "active surfaces" produced by the immobilization of polymer/protein nanoreactors onto a solid support, enabling the selective and sensitive detection of sugar alcohols. 53 These specialized nanoreactors were constructed by encapsulating ribitol dehydrogenase within polymersomes and embedding GlpF into membrane.…”

“… OmpF underwent point mutation to introduce cysteine residues at locations inside the channel (OmpF-M), to which Gala3 peptides, undergoing reversible pH-sensitive conformational alterations, were bound, forming biovalves that acted as “gates” reversibly from an open to a closed state for controlling the in situ activity within compartments. Escherichia coli glycerol facilitator (GlpF) is a channel protein for selectively transporting sugar alcohols, demonstrating a high degree of stereoselectivity in this function . Additionally, they incorporated “active surfaces” produced by the immobilization of polymer/protein nanoreactors onto a solid support, enabling the selective and sensitive detection of sugar alcohols .…”